Methods for treating hair loss disorders

ABSTRACT

The invention provides for methods for treating a hair loss disorder in a subject by administering a Janus Kinase/Signal Transducers and Activators of Transcription inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 17/714,964, filed Apr. 6, 2022; which is a continuation of U.S. patent application Ser. No. 16/299,382, filed Mar. 12, 2019, now U.S. Pat. No. 11,298,570; which is a continuation of U.S. patent application Ser. No. 15/668,814, filed Aug. 4, 2017, now U.S. Pat. No. 10,265,258; which is a continuation of U.S. patent application Ser. No. 14/821,623, filed Aug. 7, 2015, now U.S. Pat. No. 9,895,301; which is a continuation of U.S. patent application Ser. No. 13/886,252, filed May 2, 2013, now U.S. Pat. No. 9,198,911; which is a continuation-in-part of PCT Application No. PCT/US2011/059029, filed Nov. 2, 2011, which claims the benefit of and priority to U.S. Provisional Application Ser. No. 61/409,509, filed Nov. 2, 2010, and is also a continuation-in-part of PCT Application No. PCT/US2013/034688, filed Mar. 29, 2013, which claims the benefit of and priority to U.S. Provisional Patent Application Ser. No. 61/617,225, filed on Mar. 29, 2012, and U.S. Provisional Patent Application Ser. No. 61/645,499, filed on May 10, 2012, and the contents of each of the above-listed applications are hereby incorporated by reference in their entireties.

All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application.

This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

GOVERNMENT INTERESTS

This invention was made with government support under AR056016 and AR061881 awarded by the National Institutes of Health. The Government has certain rights in the invention.

SEQUENCE LISTING

The specification further incorporates by reference the Sequence Listing submitted herewith via EFS on Jul. 19, 2022. Pursuant to 37 C.F.R. § 1.52(e)(5), the Sequence Listing file, identified as 070050_6642.xml, is 162,222 bytes and was created on Jul. 19, 2022. The Sequence Listing, electronically filed herewith, does not extend beyond the scope of the specification and thus does not contain new matter.

BACKGROUND OF THE INVENTION

Alopecia Areata (AA) is one of the most highly prevalent autoimmune diseases affecting over 5 million individuals in the US, and as many as 140 million worldwide AA leads to hair loss due to the collapse of immune privilege of the hair follicle and subsequent autoimmune destruction. AA is a skin disease which leads to hair loss on the scalp and elsewhere. In some severe cases, it can progress to complete loss of hair on the head or body. Although Alopecia Areata is believed to be caused by autoimmunity, the gene level diagnosis and rationally targeted therapeutics have not been developed. The genetic basis of AA is largely unknown. Its psychological impact on affected patients is devastating, particularly in children.

SUMMARY OF THE INVENTION

An aspect of the invention encompasses a method of treating a hair-loss disorder in a mammalian subject in need thereof, the method comprising administering to the subject an inhibitor of a protein tyrosine kinase (PTK) involved in cytokine signaling. In one embodiment, the inhibitor is a Jak1, Jak2, and/or a Jak3 inhibitor. In one embodiment, the inhibitor is a Stat1 and/or a Stat2 inhibitor. In a further embodiment, the inhibitor is INCB 018424. In one embodiment, the inhibitor is tofacitinib (CP690550). In another embodiment, the hair-loss disorder comprises androgenetic alopecia, telogen effluvium, alopecia areata, telogen effluvium, tinea capitis, alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex, or alopecia universalis. In one embodiment, the method further comprises determining whether the inhibitor administered induced hair growth in the subject afflicted with a hair loss disorder as compared to the subject's hair growth prior to treatment with the inhibitor. In one embodiment, the inhibitor is an antisense RNA that specifically inhibits expression of the gene that encodes the Jak1 or Jak2 protein; a siRNA that specifically targets the gene that encodes the Jak1 or Jak2 protein; or a small molecule. In one embodiment, the inhibitor is an antibody that specifically binds to a Jak3 protein or a fragment thereof; an antisense RNA or antisense DNA that decreases expression of the gene that encodes the Jak3 protein; an antisense RNA or antisense DNA that decreases expression of the Jak3 protein; a siRNA that specifically targets the Jak3 gene; a small molecule; or a combination thereof. In one embodiment, the small molecule is Janex 1 (WHI-P131), PF-956980, WHI-P154, tofacitinib (CP690550), VX-509, JAK3 Inhibitor IV, NSC114792, or R348. In some embodiments, the antibody specifically binds to a protein comprising SEQ ID NO: 109. In another embodiment, the siRNA is directed to a human nucleic acid sequence comprising SEQ ID NO: 110. In some embodiments, the siRNA directed to a Jak3 gene is any one of the sequences listed in Table 3. In another embodiment, the inhibitor is an antisense RNA that specifically inhibits expression of the gene that encodes the Stat1 or Stat2 protein; a siRNA that specifically targets the gene that encodes the Stat1 or Stat2 protein; or a small molecule. In one embodiment, the small molecule is AG490, CYT387, SB1518, LY3009104, TG101348, BMS-911543, or CEP-701. In another embodiment, the small molecule is WP-1034 (Faderl et al., Anticancer Res. 2005 May-June; 25(3B):1841-50), fludarabine (Fludara, Berlex, Calif.), epigallocatechin-3-gallate (EGCG), or Hyperforin. In another embodiment, the siRNA is directed to a human nucleic acid sequence comprising SEQ ID NOS: 2, 4, 6 or 8. In some embodiments, the siRNA directed to a Jak1 gene is any one of the sequences listed in Table 1. In other embodiments, the siRNA directed to a Stat1 gene is any one of the sequences listed in Table 2. In a further embodiment, the administering comprises a subcutaneous, intra-muscular, intra-peritoneal, or intravenous injection; an infusion; oral, nasal, or topical delivery; or a combination thereof. In some embodiments, the administering occurs daily, weekly, twice weekly, monthly, twice monthly, or yearly. In some embodiments, the inhibitor, e.g., a Jak1, Jak2, and/or Jak3 inhibitor, is administered 1 time per week, 2 times per week, 3 times per week, 4 times per week, 5 times per week, 6 times per week, 7 times per week, 8 times per week, 9 times per week, 10 times per week, 9 times per week, 10 times per week, 11 times per week, 12 times per week, 13 times per week, or 14 times per week. In other embodiments, the subject is administered the inhibitor for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 8 weeks, at least 12 weeks, or at least 16 weeks. In some embodiments, the method comprises administering a Jak1/2 inhibitor and a Jak2 inhibitor to the subject. In further embodiments, administering the Jak1/2 inhibitor is conducted simultaneously with the administering of the Jak3 inhibitor. Yet in other embodiments, administering the Jak1/2 inhibitor is conducted sequentially in any order with the administering of the Jak3 inhibitor. In some embodiments, the Jak1/2 inhibitor is INCB 018424, GLPG0634, AG490, CYT387, SB1518, LY3009104 (Baricitinib; INCB28050), AZD1480, TG101348, BMS-911543, or CEP-701.

An aspect of the invention provides for a method for inducing hair growth in a subject where the method comprises administering to the subject an effective amount of an inhibitor of a protein tyrosine kinase (PTK) involved in cytokine signaling. In one embodiment, the inhibitor is a Jak1, Jak2, Jak3, and/or Stat inhibitor. In other embodiments, the inhibitor is INCB 018424. In one embodiment, the inhibitor is a Jak3 inhibitor. In one embodiment, the inhibitor is tofacitinib (CP690550). In some embodiments, the subject is afflicted with a hair-loss disorder. In other embodiments, the hair-loss disorder comprises androgenetic alopecia, telogen effluvium, alopecia areata, telogen effluvium, tinea capitis, alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex, or alopecia universalis. In some embodiments, the modulating compound can also inhibit hair growth, thus it can be used for treatment of hair growth disorders, such as hypertrichosis. In one embodiment, the method further comprises the step (b) determining whether the inhibitor administered induced hair growth in the subject afflicted with a hair loss disorder as compared to the subject's hair growth prior to treatment with the inhibitor. In one embodiment, the inhibitor is an antisense RNA that specifically inhibits expression of the gene that encodes the Jak1 or Jak2 protein; a siRNA that specifically targets the gene that encodes the Jak1 or Jak2 protein; or a small molecule. In one embodiment, the inhibitor is an antibody that specifically binds to a Jak3 protein or a fragment thereof, an antisense RNA or antisense DNA that decreases expression of the gene that encodes the Jak3 protein; an antisense RNA or antisense DNA that decreases expression of the Jak3 protein; a siRNA that specifically targets the Jak3 gene; a small molecule; or a combination thereof. In one embodiment, the small molecule is Janex 1 (WHI-P131), PF-956980, WHI-P154, tofacitinib (CP690550), VX-509, JAK3 Inhibitor IV, NSC114792, or R348. In some embodiments, the antibody specifically binds to a protein comprising SEQ ID NO: 109. In another embodiment, the siRNA is directed to a human nucleic acid sequence comprising SEQ ID NO: 110. In some embodiments, the siRNA directed to a Jak3 gene is any one of the sequences listed in Table 3. In another embodiment, the inhibitor is an antisense RNA that specifically inhibits expression of the gene that encodes the Stat1 or Stat2 protein; a siRNA that specifically targets the gene that encodes the Stat1 or Stat2 protein; or a small molecule. In one embodiment, the small molecule is AG490, CYT387, SB1518, LY3009104, TG101348, BMS-911543, or CEP-701. In another embodiment, the small molecule is WP-1034 (Faderl et al., Anticancer Res. 2005 May-June; 25(3B):1841-50), fludarabine (Fludara, Berlex, Calif.), epigallocatechin-3-gallate (EGCG), or Hyperforin. In another embodiment, the siRNA is directed to a human nucleic acid sequence comprising SEQ ID NOS: 2, 4, 6 or 8. In some embodiments, the siRNA directed to a Jak1 gene is any one of the sequences listed in Table 1. In other embodiments, the siRNA directed to a Stat1 gene is any one of the sequences listed in Table 2. In a further embodiment, the administering comprises a subcutaneous, intra-muscular, intra-peritoneal, or intravenous injection; an infusion; oral, nasal, or topical delivery; or a combination thereof. In some embodiments, the administering occurs daily, weekly, twice weekly, monthly, twice monthly, or yearly. In some embodiments, the inhibitor, e.g., a Jak1, Jak2, and/or Jak3 inhibitor, is administered 1 time per week, 2 times per week, 3 times per week, 4 times per week, 5 times per week, 6 times per week, 7 times per week, 8 times per week, 9 times per week, 10 times per week, 9 times per week, 10 times per week, 11 times per week, 12 times per week, 13 times per week, or 14 times per week. In other embodiments, the subject is administered the inhibitor for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 8 weeks, at least 12 weeks, or at least 16 weeks. In some embodiments, the method comprises administering a Jak1/2 inhibitor and a Jak2 inhibitor to the subject. In further embodiments, administering the Jak1/2 inhibitor is conducted simultaneously with the administering of the Jak3 inhibitor. Yet in other embodiments, administering the Jak1/2 inhibitor is conducted sequentially in any order with the administering of the Jak3 inhibitor. In some embodiments, the Jak1/2 inhibitor is INCB 018424, GLPG0634, AG490, CYT387, SB1518, LY3009104 (Baricitinib; INCB28050), AZD1480, TG101348, BMS-911543, or CEP-701.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D are photographic images of clinical manifestations of AA. In the upper panels (FIGS. 1A-1B), patients with AA multiplex. In FIG. 1B, the patient is in regrowth phase. For patients with alopecia universalis, there is a complete lack of body hair and scalp hair (FIG. 1C), while patients with alopecia totalis only lack scalp hair (FIG. 1D). In FIG. 1D, hair regrowth is observed in the parietal region, while no regrowth in either occipital or temporal regions is evident.

FIG. 2 shows a diagram showing the inflammatory response underlying Alopecia Areata (AA).

FIG. 3 shows a diagram showing the inflammatory response underlying Alopecia Areata (AA).

FIG. 4A is a photograph of lymph nodes and spleen isolated from C3H mice afflicted with AA (top) and control mice (bottom).

FIG. 4B show the results from FACS analysis that NKG2D bearing CD8 and Th1 cells are expanded in murine AA cutaneous lymph nodes.

FIG. 4C is a bar graph that shows a 6-fold increase in the number of cytokine producing cells in C3H mice afflicted with AA as compared to control mice.

FIG. 5A is a RNA signature of alopecic skin in C3H mice. The heat map shows that 16 of the top 20 unregulated genes are Interferon-Response Genes. 16 of top 20 upregulated genes are Interferon Responsive Genes (IFN-γ but not Type I IFNs are elevated)

FIG. 5B shows transcriptional profiling of AA skin. qPCR verification of single genes picked from the list generated shows an average fold change over 3 experiments.

FIG. 6 is a diagram showing the targets in AA.

FIG. 7 shows a diagram showing the interferon γ pathway.

FIGS. 8A-8B show two gene mapping approaches. FIG. 8A shows linkage analysis in families. FIG. 8B shows association analysis in a population cohort.

FIG. 9 shows a Genomewide Association Study (GWAS) in AA.

FIGS. 10A-10B show danger signals: ULBP3 expression in the hair follicle in both control individuals (FIG. 10A) and AA patients (FIG. 10B).

FIGS. 11A-11B show that AA is caused by a “swarm of bees”: Killer CD8 T cells attracted by NKG2DL. FIG. 11A shows “pre-2010” swarm of bees in AA. FIG. 11B shows “2012” identification of bees ((m) staining of CD8; (n) staining of NKG2D; and (o) colocalization of NKG2D and CD8).

FIG. 12 shows a diagram that shows the inflammatory response underlying AA.

FIG. 13 shows INCB18424-202: Th1 and Th17 transcript markers and keratin 16 are decreased with topical INCB18424, a JAK1 and JAK2 inhibitor, treatment.

FIG. 14 shows the Key Total Lesion Score results for INCB18424-203. The results how that Total Lesion Score for all dose groups ≥2× decrease over vehicle, achieving significance after control for multiplicity

FIGS. 15A-15B show that treatment with INCB018424 markedly reduces epidermal thickening and dermal inflammation. No pathological skin thinning is observed.

FIG. 16 is a schematic that shows the pre-clinical strategies to interrupt AA pathogenesis are aimed at interruption of the induction and effector function of CD8 T cells and include blocking IFN-γ signals.

FIG. 17 shows that CD8+ NKG2D+ T cells infiltrate AA hair follicles in both mouse and human. Top panel: Human Alopecia Areata (Pethukova et al., 2010, Nature, 466(7302):113-7); bottom panel—C3H/HeJ Alopecia Areata.

FIG. 18-1 , FIG. 18-2 , and FIG. 18-3 show CD8+ NKG2D+ T cells in alopecic C3H mice CD8□αβ⁺ NKG2D⁺ T cells are massively expanded in the skin, lymph node and blood of alopecic C3H mice. (NKG2D⁺ γδ T cells are present in both affected and unaffected skin).

FIG. 19 . shows CD8+ NKG2D+ T cells in alopecic C3H mice. Immunophenotype of gated LN CD8α⁺ NKG2D⁺ populations shows that they are uniformly CD8αβ⁺ CD44⁺ CXCR3⁺ DX5⁺ NKG2A/C/E+ IFN-γ producing memory T cells.

FIG. 20 shows spectratype analysis of C3H T cells in skin and LN: Similar TCR repertoires are shared by total skin and sorted CD8+ NKG2D+LN T cells identifying these as the relevant effectors.

FIGS. 21A-21B show that IFN-γ producing CD8 T cells dominate human and mouse AA. FIG. 21A: Human: T cells obtained from T cell crawl-outs from AA skin biopsies are uniformly CD8+ NKG2D+ and produce IFN-γ upon PMA/Ionomycin stimulation. FIG. 21B: Mouse: C3H/HeJ stimulated lymph node cells from alopecic mice contain 3 times more IFN-γ producing CD8 and CD4 T cells than unaffected mice.

FIG. 22 shows that Type I and II IFNs are upregulated in C3H mouse AA skin: fold changes in expression of interferon genes (normalized to 18S) between 3 affected and 3 non affected C3H/HeJ mice, utilizing an interferon signaling and response qPCR array (Stellarray Lonza Cat #00189608).

FIG. 23 shows graphs that show elevated serum chemokines and cytokines in Human AA. Interferon-y and IFN-induced chemokines (e.g., IP-10/CXCL10) are elevated in the serum of human AA, in some cases correlating with disease severity, i.e., patchy disease (AAP) vs. universalis (AU).

FIG. 24 is a luminex analysis that shows elevated IL-13 in the serum of Human AA subjects, in some cases correlating with disease severity, i.e., patchy disease (AAP) vs. universalis (AU).

FIG. 25 shows flow cytometry data that were gathered on a LSRII using the following mAbs in one tube to stain the entire population of C3H dermal cells liberated from a 0.5 cm by 2 cm patch of alopecic skin after 30 minutes collagenase digestion: (MHC II, CD11b, CD11c, CD19, 120G8, CD3, CD4, CD8, DX5, NKG2D). γδNKG2D positive cells and CD8+DX5+ NKG2D+ population are identified. These cells were also CD3+, with γδ□CD3 expression 100× higher than that seen in CD8+NK+ T cells. >95% of the total CD3 positive cells were accounted.

FIG. 26 is a graph that shows the evaluation of PBMCs from four AA subjects. NKG2D expression was increased in subsets of lymphocytes.

FIG. 27 shows spectratyping in a C3H-HeJ mouse. RNA was Isolated from skin biopsies, and from sorted NKG2D− and NKG2D+CD8+ T cells from cutaneous lymph nodes of C3H-HeJ mice. Spectratyping analysis shows CDR3 length skewing in several Vbeta families. Some of the skewed Vbeta families are shared between skin and NKG2D+ lymph node CD8+ T cell populations (e.g., Vbeta2, Vbeta5.2, Vbeta20) whereas additional Vbeta families are skewed in skin (reflecting the CD4+ T cell population).

FIG. 28 is a schematic showing that human scalp skin procured from control or AA affected individuals can be used to establish primary cultures of individual cellular populations within the skin and hair follicle.

FIGS. 29A-29D show a T cell ‘crawl-out’ assay, in which cultured human dermis obtained from punch skin biopsies are used as source material for flow cytometric analysis. Trisected skin punch biopsies of a 6 mm skin biopsy is obtained from alopecic lesional and non-lesional areas. After removing the fat, the finely minced skin is gently pressed in a Cellfoam matrix and then transferred, skin side-up, to a well of a 24-well plate containing 2 mls of Skin T medium with IL-2 (6 ng/ml) and IL-15 (10 ng/ml). T cells crawl outs are shown in FIG. 29A after the matrix is removed 3 days later. Flow cytometry (FIGS. 29B-29C) and spectratyping (FIG. 29D) of these cells are also shown.

FIG. 30 shows that human peripheral blood mononuclear cells (PBMC), top panels or murine splenocytes were cultured in high dose IL-2 (1000/ml) to generate lymphokine-activated killer (LAK) cells. The CFSE green fluorescence labeled LAK cells were incubated with human hair follicle organ culture (top panels), or mouse hair follicle culture (bottom panels) from non-lesion region (bottom left two) or lesion.

FIG. 31 shows immunohistochemical (IHC) staining of unaffected and affected AA skin in C3H-HeJ mice.

FIG. 32 shows that treatment of mice with the Jak3 inhibitor (tofacitinib; CP-69055) prevents alopecia areata (AA).

FIGS. 33A, 33B-1, AND 33B-2 show treatment related elimination of dermal T cell infiltrates and inflammatory biomarkers by immunostaining (FIG. 33A) and by flow cytometry (FIG. 33B-1 -FIG. 33B-2 ). CD8+ NKG2D+ T cells, which represent up to 25% of dermal cells in AA skin, are completely eliminated with treatment. The flow cytometry is of single cell suspensions from the skin. The cells in the right upper quadrant are not observed in normal healthy mice and are CD8+ NKG2D+ effector T cells. CD8+ NKG2D+ effector T cells are found in alopecic mice after skin grafting. Mice treated with the JAK3 inhibitor do not develop alopecia and their skin is devoid of these cells.

FIG. 34 are graphs that show treatment elimination of IFN-7 producing “NK-type” CD8 T cells in cutaneous LN, and loss of circulating IFN-inducible chemokines. The bar graphs in the left panel show intracellular flow cytometry of PMA/Iono stimulated LN cells from individually treated and untreated mice stained for CD8.NKG2A/C/D/E.IFNγ. Two mice per group are shown.

FIG. 35 is a ribbon diagram that shows C3H/HeJ graft recipients were treated with a JAK3 inhibitor (right panel), or PBS. RNA was extracted from skin harvested at weeks 6 (w6) and 13 (w13) following treatment and also from mice that had undergone sham surgery and subsequently interrogated by microarray analysis.

FIG. 36 shows photographs of mice before (top panel) and after topical treatment (9 days (middle panel) and 30 days (bottom panel)) with cream only.

FIG. 37 shows photographs of mice before (top panel) and after topical treatment (9 days (middle panel) and 30 days (bottom panel)) with the Jak3 inhibitor, tofacitinib.

FIG. 38 shows photographs of mice before (top panel) and after a control infusion (5 weeks (middle panel) and 10 weeks (bottom panel)) without the Jak3 inhibitor, tofacitinib.

FIG. 39 shows photographs of mice before (top panel) and after a pump infusion (5 weeks (middle panel) and 10 weeks (bottom panel)) with the Jak3 inhibitor, tofacitinib.

FIG. 40 shows treatment related elimination of dermal T cell infiltrates and inflammatory biomarkers by flow cytometry. The flow cytometry is of lymph node cells. The cells in the right upper quadrant are not observed in normal healthy mice and represent CD8+ NKG2D+ effector T cells. CD8+ NKG2D+ effector T cells are found in alopecic mice after skin grafting. Alopecic mice treated with the JAK3 inhibitor exhibit hair regrowth and their lymph nodes are devoid of these cells.

FIG. 41 shows that NKG2D “stress ligands” are upregulated in the hair follicle, and demonstrates the shared pathogenesis in human and mouse.

FIG. 42 is a photomicrograph of an immunostain showing that CD8⁺ NKG2D⁺ T cells infiltrate the hair follicle, and demonstrates the shared pathogenesis in human and mouse.

FIG. 43 shows flow cytometric analysis that shows that NKG2D⁺ CD8⁺ T cells are the dominant infiltrating cells, and demonstrates the shared pathogenesis in human and mouse. (e.g., see Clark, et al., (2006) J Invest Dermatol. 126(5):1059-70, reporting a method for isolating T cell crawlouts from the skin).

FIG. 44 shows that comparative transcriptomics reveals a shared IFN response signature, and demonstrates shared pathogenesis in human and mouse.

FIG. 45 shows plots depicting that CD8+ NKG2D+ Cytotoxic T Cells are IFN-gamma producing cells bearing NK-type receptors.

FIG. 46 shows that NK Type CD8 T cells are required for T Cell transfer of Alopecia Areata.

FIG. 47 shows pre-clinical studies using systemic JAK inhibitors in the C3H-HeJ mouse model of alopecia areata (for example, see O'Shea, Immunity. 2012 Apr. 20; 36(4):542-50).

FIG. 48 shows the results of pre-clinical studies with systemic delivery of JAK3 inhibitor (tofacitinib), and tofacitinib treatment prevents Alopecia Areata.

FIG. 49 is a diagram depicting different examples of targeted therapies for alopecia areata. These topical therapies can be used to reverse the established disease.

FIG. 50 shows pre-clinical studies with topical delivery of JAK3 inhibitor (tofacitinib). Topical treatment using JAK3 inhibitor results in reversal of long-standing AA (2-3 months duration). Before treatment, all mice had hair loss for 2-3 months. Control mice were applied with cream daily. Mice treated with the JAK3 inhibitor, Tofacitnib, were treated daily with a topical cream containing 0.5% Tofacitnib.

FIG. 51 shows a summary of pre-clinical studies in alopecia C3H/HeJ mice.

FIG. 52 shows the prevention of AA with systemic treatment with a JAK3 inhibitor given at the time of skin grafting. All reagents are started at time of graft (prevention). JAK3 inhibitor (CP690550) was delivered using Alzet osmotic mini-pumps (28-day pumps, Model 2004) implanted subcutaneously, 10 mg/kg/day or vehicle (PEG300).

FIG. 53 shows photomicrographs depicting that treatment with Jak3 inhibitor tofacitinib normalizes inflammatory infiltrate.

FIG. 54A shows the upregulation of NKG2DL in the hair follicle of C3H/HeJ alopecic mice. Immunofluorescence staining of NKG2D ligands (H60) in the hair follicle inner root sheath (marked by K71).

FIG. 54B shows the cellular phenotype and function of CD8⁺ NKG2D⁺ cytotoxic T lymphocytes in AA mice. Immunofluorescence staining of CD8⁺ NKG2D⁺ cells in hair follicles of C57BL/6, healthy C3H/HeJ and C3H/HeJ AA mice.

FIG. 54C shows the cellular phenotype and function of CD8+ NKG2D+ cytotoxic T lymphocytes in AA mice. Striking cutaneous lymphadenopathy and cellularity in C3H/HeJ mice that developed AA.

FIG. 54D shows the cellular phenotype and function of CD8⁺ NKG2D⁺ cytotoxic T lymphocytes in AA mice. Plots show the frequency of CD8⁺ NKG2D⁺ T cells from skin and skin-draining lymph nodes in alopecic mice vs. ungrafted mice.

FIG. 54E shows the cellular phenotype and function of CD8⁺ NKG2D⁺ cytotoxic T lymphocytes in AA mice. Plots shows the immunophenotype of CD8⁺ NKG2D⁺ T cells in cutaneous lymph nodes in C3H/HeJ alopecic mice.

FIG. 54F shows the cellular phenotype and function of CD8⁺ NKG2D⁺ cytotoxic T lymphocytes in AA mice. Graphs demonstrate that CD8⁺ NKG2D⁺ T cells isolated from AA mice cutaneous LNs kill Rae-1 expressing dermal sheath cells grown from C3H/HeJ hair follicles in an NKG2D-dependent manner.

FIG. 54G shows the cellular phenotype and function of CD8⁺ NKG2D⁺ cytotoxic T lymphocytes in AA mice. RNAseq was performed on CD8+ NKG2D+ and CD8+ NKG2D− T cells sort purified from AA cutaneous lymph nodes. Transcripts upregulated in CD8+ NKG2D+ T cells were compared with CTL gene expression and NK cell gene expression in the literature^(8,9), and overlapping gene signatures are displayed in this Venn Diagram.

FIG. 54H shows the cellular phenotype and function of CD8⁺ NKG2D⁺ cytotoxic T lymphocytes in AA mice. C3H/HeJ mice were injected subcutaneously with 2×10⁶ cells from four different populations. Hair loss developed in recipients after either injection of total LN cells or CD8⁺ NKG2D⁺ T cells alone (5 out of 5 mice per group), whereas mice receiving CD8⁺ NKG2D⁻ T cells or LN cells depleted of NKG2D+ cells did not develop alopecia (0 out of 5 mice per group). ***p value=<0.001.

FIG. 55A shows transcriptional profiling of human and mouse AA skin that reveals evidence for IFNγ, cytotoxic T cells and the IL15 pathway as central to disease pathogenesis, inviting therapeutic targeting using Jak inhibitors. FIG. 55A is a Venn diagram showing overlap of gene list from whole alopecic C3H/HeJ mouse skin (compared with unaffected C3H/HeJ skin) with IFNγ-induced gene expression¹¹ and CD8 and NK cell gene signatures from ImmGen consortium publications^(8,9) (Top panel). Representative list of differentially expressed genes among human AA and C3H/HeJ AA reveals shared inflammatory pathways, in particular IFNγ pathway genes, genes representative of CD8 effectors, and a striking IL-15 pathway signature (Bottom panel).

FIGS. 55B-55D show effect of treatment with a Jak3 inhibitor (JAK3i). FIG. 55B, JAK3i (tofacitinib) inhibits IL-15-induced Stat3 activation in T cells. FIG. 55C, JAK3i inhibits IL-15-induced LAK cell cytotoxic function. FIG. 55D, JAK3i inhibits IL-15-induced LAK cell Granzyme B expression and IFNγ production.

FIG. 56A shows targeting CD8⁺ NKG2D⁺ cytotoxic T lymphocytes with JAK3i prevents the onset of alopecia in grafted C3H/HeJ mice. C3H/HeJ grafted mice were treated systemically from the time of grafting. The onset of alopecia is inhibited by JAK3i, tofacitinib treatment. The durability of disease prevention was demonstrated by treatment withdrawal for an additional 12 weeks after treatment withdrawal.

FIG. 56B shows targeting CD8⁺ NKG2D⁺ cytotoxic T lymphocytes with JAK3i prevents the onset of alopecia in grafted C3H/HeJ mice. C3H/HeJ grafted mice were treated systemically from the time of grafting. The onset of alopecia is inhibited by JAK3i, tofacitinib treatment.

FIG. 56C shows targeting CD8⁺ NKG2D⁺ cytotoxic T lymphocytes with JAK3 prevents the onset of alopecia in grafted C3H/HeJ mice. The frequency of CD8⁺ NKG2D⁺ T cells in skin and cutaneous lymph nodes of JAK3i treated mice were significantly decreased compared to control mice. ***p value=<0.001.

FIG. 56D shows targeting CD8⁺ NKG2D⁺ cytotoxic T lymphocytes with JAK3i prevents the onset of alopecia in grafted C3H/HeJ mice. Immunohistochemical staining of skin biopsies showed that the expression of CD4, CD8, MHC class I and II in skin are significantly decreased in JAK3i treated mice compared to control mice.

FIG. 56E shows targeting CD8⁺ NKG2D⁺ cytotoxic T lymphocytes with JAK3i prevents the onset of alopecia in grafted C3H/HeJ mice. ALADIN score (a summary statistic of expression of IFN signature and CTL signature genes) from JAK3i treated mice showed normalization. For gene expression studies, mice grafted with autologous health skin were included as sham-operated controls.

FIG. 56F shows targeting CD8⁺ NKG2D⁺ cytotoxic T lymphocytes with JAK3i prevents the onset of alopecia in grafted C3H/HeJ mice. GEDI results of JAK3i treated mice showed reduction in CTL and IFN signatures.

FIG. 57A shows reversal of established AA with topical small molecule inhibitor of the downstream effector kinase JAK3. Three-to-five per group of mice with long-standing AA (at least 12 weeks after grafting) were treated topically on the dorsal back with 0.5% JAK3i, tofacitinib or (top panels) vehicle alone (AQUAPHOR™) by daily application for 12 weeks. A full coat of hair emerged in the JAK3i treated mice by 7 weeks of treatment and further developed by 12 weeks. The durability of hair regrowth was measured for an additional 8 weeks after treatment withdrawal with no evidence of disease recurrence.

FIG. 57B shows reversal of established AA with topical small molecule inhibitor of the downstream effector kinase JAK3. The graph depicts a time course of hair regrowth index shown as weeks after treatment.

FIG. 57C shows reversal of established AA with topical small molecule inhibitor of the downstream effector kinase, JAK3. The plots depict the frequency of CD8⁺ NKG2D⁺ T cells in the skin of JAK3i-treated mice was significantly decreased compared to control mice. *p value=<0.05, **p value=<0.01, n.s.=not significant.

FIG. 57D shows reversal of established AA with topical small molecule inhibitor of the downstream effector kinase, JAK3. Immunohistochemical staining of skin biopsies shows treatment-related loss of expression of CD4, CD8, MHC class I and II markers.

FIG. 57E shows reversal of established AA with topical small molecule inhibitor of the downstream effector kinase, JAK3. The ALADIN score shows treatment-related loss of CTL and IFN signatures.

FIG. 57F shows reversal of established AA with topical small molecule inhibitor of the downstream effector kinase, JAK3. GEDI analysis shows territories of gene expression with treatment-related reversal of pathogenic signatures.

FIG. 58A shows that Rae-1 is upregulated in the AA HF. Immunostaining of lesional using a pan-Rae-1 antibody in C3H/HeJ alopecic mice, unaffected C3H/HeJ mice and C57Bl.6 mice.

FIG. 58B shows that H60 and Rae-1 are overexpressed in AA. NKG2DL RNA expression in alopecic lesional skin from C3H/HeJ mice compared with non-lesional skin from unaffected C3H/HeJ mice. RT-PCR data from cDNA from 3 mice are shown and represented as relative fold-induction.

FIG. 58C shows that CD4 T cells are infrequent in AA lesional skin: Flow cytometric evaluation of lesional alopecic skin. Quantitation of CD4 and CD8 T cells as a percentage of total gated CD3⁺ or CD45⁺ cells.

FIG. 58D shows that effector memory immunophenotype of CD8⁺ NKG2D⁺ T cells are similar in lesional skin and in the cutaneous draining lymph node. Gated CD8alpha⁺ NKG2D⁺ T cells are displayed for CD8beta, NKG2D, NKG2A/C/E, CD44, CD103 and CD62L expression.

FIGS. 59, 59A-59D and 59-2 show a network map of differentially expressed upregulated genes in CD3+CD8⁺ NKG2D+LN cells vs. CD8⁺ NKG2D− cells. String.db was used to create a biological interaction score matrix with the differentially expressed genes. The network map was created using cytoscape; only biological interactions >0.75 were used. Nodes represent genes, and edges represent biological interactions as derived from string.db. Node size is proportional to fold change, and edge width is proportional to biological interaction.

FIGS. 60A-60B show validation of mouse RNA expression studies. To determine the expression signature of C3H/HeJ mouse skin affected with alopecia areata, lesional skin was isolated from three affected female mice and three unaffected aged-matched controls. Total and small RNAs were isolated from whole skin and biotin-labeled cRNA was generated through in vitro transcription, followed by hybridization to the Affymetrix Mouse 430 2.0 Genechip. Data analysis was done as outlined in the Methods. FIG. 60A is a heatmap depicting the significantly and differentially expressed genes between C3H/HeJ affected and unaffected skin. FIG. 60B shows validation by qRT-PCR of several selected immune-related genes from this list whose expression levels are significantly upregulated in AA lesional skin compared to unaffected skin, where each bar represents the average fold change of three independent experiments. UA=unaffected; AA=affected.

FIG. 61A shows systemic treatment of AA mice with JAK3 inhibitor. C3H/HeJ mice with long-standing alopecia areata were treated with tofacitinib with Alzet osmotic mini-pumps (pumps, model 2004, Durect Corporation) implanted subcutaneously on the back of each mouse to deliver vehicle (poly(ethylene glycol) (PEG)300) or vehicle containing JAK3i tofacitinib (Abmole) at 15 mg/kg/day for 12 wks. Alopecia areata reversal was complete on both the back and belly.

FIG. 61B shows systemic treatment of AA mice with JAK3 inhibitor. Flow cytometric analysis of skin and cutaneous lymph node populations shows elimination of the CD8⁺ NKG2D⁺ T cell population in treated mice (n=3 per group).

FIG. 61C shows systemic treatment of AA mice with JAK3 inhibitor. Immunostaining of skin from mice treated with tofactinib or placebo demonstrates elimination of CD8 infiltration and MHC I and II upregulation in tofacitinib treated mice.

FIG. 62 depicts JAK-STAT signalingin normal hair follicle development. To analyze the status of the JAK-STAT signaling pathway during a normal hair cycle, mRNA was collected from skin of three mice at postnatal days 17, 23, 29, 33. Samples represent the telogen to anagen transition as evident in the photomicrographs of H&E stains from these time points. Day 17=early telogen; day 23=late telogen; day 29=early anagen; day 33=full anagen. cDNA was made and probed on JAK-STAT qPCR arrays. JAK-STAT signaling is up in telogen and down in anagen (see FIG. 63 ).

FIG. 63 is a heat map showing a hierarchical cluster analysis. mRNA isolated from mouse skin at each time point (D17, 23, 29, 33) were probed on JAK-STAT qPCR array. Red=induced, Green=repressed. Shown are all genes of the array. Several clusters of genes are evident that are upregulated in both telogen time points (d17 and d23), or upregulated in both anagen time points (d29 and d33).

FIG. 64A is a heat map showing a hierarchical cluster analysis. The figure shows averages of 3 biological replicates/time point. The Control group: D17 (Telogen); Group 1: D23 (telogen); Group 2: D29 (anagen); and Group 3: D33 (anagen). JAK1 and 3 are upregulated in telogen group (red in control group and Group 1). Expression of JAK-STAT signaling pathway components is up in telogen and down in anagen.

FIG. 64B is a heat map showing a hierarchical cluster analysis. The figure shows averages of 3 biological replicates/time point. The Control group: D17 (Telogen); Group 1: D23 (telogen); Group 2: D29 (anagen); and Group 3: D33 (anagen). Stat 1/2/3/5 are upregulated in telogen (red in control group and group 1). Expression of JAK-STAT signaling pathway components is up in telogen and down in anagen.

FIG. 65 is a heat map (top) showing a cluster analysis. The Control group: D17 (Telogen); Group 1: D23 (telogen); Group 2: D29 (anagen); and Group 3: D33 (anagen). Potential ligands for JAK STAT or targets for JAK3i: OSM, IL6st (GP130), IL-4, TL-2Rg, CSF1R, and others upregulated in telogen samples.

FIGS. 66-1-66-2 show summaries of genes upregulated in telogen as well as downregulated in telogen. The Control group: D17 (Telogen); Group 1: D23 (telogen); Group 2: D29 (anagen); and Group 3: D33 (anagen). CRP, a well-defined gene upregulated by JAK/STAT signaling, is strikingly increased in telogen.

FIG. 67 are photomicrographs showing JAK-STAT in hair follicle development: Embryonic development. Stat 3 and Stat 5 were identified to be differentially expressed between telogen and anagen. To examine the pattern of expression in embryonic development, the activated (phosphorylated) forms of these proteins were stained. As shown, P-Stat 3 is expressed in epithelial layers during early stages of development and then can be faintly seen in the dermal papilla. P-Stat5 expression appears later, at E16.5, in scattered dermal cells and becomes pronounced in the dermal condensate by E18.5.

FIG. 68 are photomicrographs showing JAK-STAT in hair follicle development: post-natal development. P-Stat3 is expressed in the basal layers of the epidermis and upper epithelial layers of the follicle during neonatal skin development. Expression can also be detected in the dermal papilla, most obviously during telogen (D17). P-Stat5 is highly expressed in the dermal papilla in all stage of the hair cycle. In telogen, expression of P-Stat5 maybe limited to a subset of DP cells, closest to the K15+ bulge (D17, bottom).

FIG. 69 are photomicrographs showing the effect of a JAK-STAT inhibitor on the hair cycle of normal mice: Induction of anagen. Inhibition of the JAK-STAT pathway during telogen (by application of a JAK inhibitor) results in early onset of anagen. 7-8 weeks old animals in telogen were treated with controls or JAK STAT inhibitors. Negative (vehicle alone; Left Panel) and positive (SAG=sonic hedgehog agonist; Middle panel) controls show that DMSO treatment alone does not induce anagen, while treatment with sonic hedgehog agonist results in early (4-7 days post treatment) induction of anagen, as expected. Right: Treatment with the Jak 3 inhibitor Tofacitinib (10 mg/mL) results in marked induction of anagen after 2 weeks or 3 weeks of treatment compared to vehicle treated mice which remain in telogen.

FIG. 70 are photomicrographs showing effects of drug treatment on keratinocyte proliferation in vivo. Tofacitinib does not appear to cause hyperpoliferation of the epidermis. Top: Krt6, a marker of keratinocyte proliferation typically expressed in the inner root sheath of the follicle. Bottom: the effects of drug treatment on proliferation of the follicle. In tofacitinib treated skin, proliferation in the secondary germ occurs later, indicating that Tofacitinib can induce anagen.

FIG. 71 are plots showing JAK-STAT in hair cycle: a drug treatment in mice. Quantification of number and thickness of hair follicles in skin treated with the Jak3 inhibitor, Tofacitinib, and a Jak1/2 inhibitor Ruxolitinib, as compared to plucked skin and vehicle-treated (DMSO). Both the numbers of follicles as well as the thickness of follicles is greater in drug treated skin than in vehicle treated mice.

FIG. 72 are photomicrographs showing the effect of Jak inhibitors on human hair follicle morphogenesis: drug treatments of human fetal scalp. Human embryonic scalp at 20 weeks was obtained and treated in vitro with 30 mg/mL of the Jak3 inhibitor, Tofacitinib, or vehicle alone. Skin was harvested and sectioned and assessed for hair follicle morphology. Hairs in treated skin appear more advanced in the first anagen of morphogenesis compared to DMSO treated scalp.

FIG. 73 shows topical treatment with a JAK inhibitor. Seven-week-old normal C57BL/6 mice were shaved in telogen and treated with 1% Jak3 inhibitor (Tofacitinib; Right panel), 1.5 mg/ml Isopropyl unoprostone (LATISSE™; Middle Right panel), 100 uM SAG (shh agonist; Middle Left panel), or DMSO as vehicle (Left Panel) by daily application for 3 weeks. Effect is durable out to a little more than 1 month at the time the image was taken. Right half of each mouse was administered topical drug, left half of each mouse was treated with DMSO alone.

FIG. 74 are photomicrographs of Ki67 staining (green) showing marked proliferation in the telogen hair follicle in Tofacitinib and SAG treated skin, compared to vehicle, indicative of the start of Anagen.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides for methods of treating a hair loss disorder (e.g., Alopecia Areata (AA), a common autoimmune form of hair loss) with an inhibitor of the Jak/Stat pathway, including Jak1, Jak2, Jak3, Stat1, and Stat2. Clinical research in AA has lagged behind its more heavily investigated “sister” autoimmune diseases in which this gene has been implicated (e.g., rheumatoid arthritis (RA), type 1 diabetes mellitus (T1D), multiple sclerosis (MS)). The invention provides for therapeutics previously untested in AA, that can inform one about the clinical relevance of Jak/Stat pathways in AA and related diseases.

Abbreviations and Definitions

The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise.

As used herein the term “about” is used herein to mean approximately, roughly, around, or in the region of When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

Overview of the Integument and Hair Cells

The integument (or skin) is the largest organ of the body and is a highly complex organ covering the external surface of the body. It merges, at various body openings, with the mucous membranes of the alimentary and other canals. The integument performs a number of essential functions such as maintaining a constant internal environment via regulating body temperature and water loss; excretion by the sweat glands; but predominantly acts as a protective barrier against the action of physical, chemical and biologic agents on deeper tissues. Skin is elastic and except for a few areas such as the soles, palms, and ears, it is loosely attached to the underlying tissue. It also varies in thickness from 0.5 mm (0.02 inches) on the eyelids (“thin skin”) to 4 mm (0.17 inches) or more on the palms and soles (“thick skin”) (Ross M H, Histology: A text and atlas, 3^(rd) edition, Williams and Wilkins, 1995: Chapter 14; Burkitt H G, et al, Wheater's Functional Histology, 3^(rd) Edition, Churchill Livingstone, 1996: Chapter 9).

The skin is composed of two layers: a) the epidermis and b) the dermis. The epidermis is the outer layer, which is comparatively thin (0.1 mm). It is several cells thick and is composed of 5 layers: the stratum germinativum, stratum spinosum, stratum granulosum, stratum lucidum (which is limited to thick skin), and the stratum corneum. The outermost epidermal layer (the stratum corneum) consists of dead cells that are constantly shed from the surface and replaced from below by a single, basal layer of cells, called the stratum germinativum. The epidermis is composed predominantly of keratinocytes, which make up over 95% of the cell population. Keratinocytes of the basal layer (stratum germinativum) are constantly dividing, and daughter cells subsequently move upwards and outwards, where they undergo a period of differentiation, and are eventually sloughed off from the surface. The remaining cell population of the epidermis includes dendritic cells such as Langerhans cells and melanocytes. The epidermis is essentially cellular and non-vascular, containing little extracellular matrix except for the layer of collagen and other proteins beneath the basal layer of keratinocytes (Ross M H, Histology: A text and atlas, 3^(rd) edition, Williams and Wilkins, 1995: Chapter 14; Burkitt H G, et al, Wheater's Functional Histology, 3^(rd) Edition, Churchill Livingstone, 1996: Chapter 9).

The dermis is the inner layer of the skin and is composed of a network of collagenous extracellular material, blood vessels, nerves, and elastic fibers. Within the dermis are hair follicles with their associated sebaceous glands (collectively known as the pilosebaceous unit) and sweat glands. The interface between the epidermis and the dermis is extremely irregular and uneven, except in thin skin. Beneath the basal epidermal cells along the epidermal-dermal interface, the specialized extracellular matrix is organized into a distinct structure called the basement membrane (Ross M H, Histology: A text and atlas, 3^(rd) edition, Williams and Wilkins, 1995: Chapter 14; Burkitt H G, et al, Wheater's Functional Histology, 3^(rd) Edition, Churchill Livingstone, 1996: Chapter 9).

The mammalian hair fiber is composed of keratinized cells and develops from the hair follicle. The hair follicle is a peg of tissue derived from a downgrowth of the epidermis, which lies immediately underneath the skin's surface. The distal part of the hair follicle is in direct continuation with the external, cutaneous epidermis. Although a small structure, the hair follicle comprises a highly organized system of recognizably different layers arranged in concentric series. Active hair follicles extend down through the dermis, the hypodermis (which is a loose layer of connective tissue), and into the fat or adipose layer (Ross M H, Histology: A text and atlas, 3^(rd) edition, Williams and Wilkins, 1995: Chapter 14; Burkitt H G, et al, Wheater's Functional Histology, 3^(rd) Edition, Churchill Livingstone, 1996: Chapter 9).

At the base of an active hair follicle lies the hair bulb. The bulb consists of a body of dermal cells, known as the dermal papilla, contained in an inverted cup of epidermal cells known as the epidermal matrix. Irrespective of follicle type, the germinative epidermal cells at the very base of this epidermal matrix produce the hair fiber, together with several supportive epidermal layers. The lowermost dermal sheath is contiguous with the papilla basal stalk, from where the sheath curves externally around all of the hair matrix epidermal layers as a thin covering of tissue. The lowermost portion of the dermal sheath then continues as a sleeve or tube for the length of the follicle (Ross M H, Histology: A text and atlas, 3^(rd) edition, Williams and Wilkins, 1995: Chapter 14; Burkitt H G, et al, Wheater's Functional Histology, 3^(rd) Edition, Churchill Livingstone, 1996: Chapter 9).

Developing skin appendages, such as hair and feather follicles, rely on the interaction between the epidermis and the dermis, the two layers of the skin. In embryonic development, a sequential exchange of information between these two layers supports a complex series of morphogenetic processes, which results in the formation of adult follicle structures. However, in contrast to general skin dermal and epidermal cells, certain hair follicle cell populations, following maturity, retain their embryonic-type interactive, inductive, and biosynthetic behaviors. These properties can be derived from the very dynamic nature of the cyclical productive follicle, wherein repeated tissue remodeling necessitates a high level of dermal-epidermal interactive communication, which is vital for embryonic development and would be desirable in other forms of tissue reconstruction.

The hair fiber is produced at the base of an active follicle at a very rapid rate. For example, follicles produce hair fibers at a rate 0.4 mm per day in the human scalp and up to 1.5 mm per day in the rat vibrissa or whiskers, which means that cell proliferation in the follicle epidermis ranks amongst the fastest in adult tissues (Malkinson F D and J T Kearn, Int J Dermatol 1978, 17:536-551). Hair grows in cycles. The anagen phase is the growth phase, wherein up to 90% of the hair follicles said to be in anagen; catagen is the involuting or regressing phase which accounts for about 1-2% of the hair follicles; and telogen is the resting or quiescent phase of the cycle, which accounts for about 10-14% of the hair follicles. The cycle's length varies on different parts of the body.

Hair follicle formation and cycling is controlled by a balance of inhibitory and stimulatory signals. The signaling cues are potentiated by growth factors that are members of the TGFβ-BMP family. A prominent antagonist of the members of the TGFβ-BMP family is follistatin. Follistatin is a secreted protein that inhibits the action of various BMPs (such as BMP-2, -4, -7, and -11) and activins by binding to said proteins, and purportedly plays a role in the development of the hair follicle (Nakamura M, et al., FASEB J, 2003, 17(3):497-9; Patel K Intl J Biochem Cell Bio, 1998, 30:1087-93; Ueno N, et al., PNAS, 1987, 84:8282-86; Nakamura T, et al., Nature, 1990, 247:836-8; Iemura S, et al., PNAS, 1998, 77:649-52; Fainsod A, et al., Mech Dev, 1997, 63:39-50; Gamer L W, et al., Dev Biol, 1999, 208:222-32).

The deeply embedded end bulb, where local dermal-epidermal interactions drive active fiber growth, is the signaling center of the hair follicle comprising a cluster of mesencgymal cells, called the dermal papilla (DP). This same region is also central to the tissue remodeling and developmental changes involved in the hair fiber's or appendage's precise alternation between growth and regression phases. The DP, a key player in these activities, appears to orchestrate the complex program of differentiation that characterizes hair fiber formation from the primitive germinative epidermal cell source (Oliver R F, J Soc Cosmet Chem, 1971, 22:741-755; Oliver R F and C A Jahoda, Biology of Wool and Hair (eds Roger et al.), 1971, Cambridge University Press:51-67; Reynolds A J and C A Jahoda, Development, 1992, 115:587-593; Reynolds A J, et al., J Invest Dermatol, 1993, 101:634-38).

The lowermost dermal sheath (DS) arises below the basal stalk of the papilla, from where it curves outwards and upwards. This dermal sheath then externally encases the layers of the epidermal hair matrix as a thin layer of tissue and continues upward for the length of the follicle. The epidermally-derived outer root sheath (ORS) also continues for the length of the follicle, which lies immediately internal to the dermal sheath in between the two layers, and forms a specialized basement membrane termed the glassy membrane. The outer root sheath constitutes little more than an epidermal monolayer in the lower follicle, but becomes increasingly thickened as it approaches the surface. The inner root sheath (IRS) forms a mold for the developing hair shaft. It comprises three parts: the Henley layer, the Huxley layer, and the cuticle, with the cuticle being the innermost portion that touches the hair shaft. The IRS cuticle layer is a single cell thick and is located adjacent to the hair fiber. It closely interdigitates with the hair fiber cuticle layer. The Huxley layer can comprise up to four cell layers. The IRS Henley layer is the single cell layer that runs adjacent to the ORS layer (Ross M H, Histology: A text and atlas, 3^(rd) edition, Williams and Wilkins, 1995: Chapter 14; Burkitt H G, et al, Wheater's Functional Histology, 3^(rd) Edition, Churchill Livingstone, 1996: Chapter 9).

Alopecia Areata

Alopecia areata (AA) is one of the most prevalent autoimmune diseases, affecting approximately 4.6 million people in the US alone, including males and females across all ethnic groups, with a lifetime risk of 1.7% (1) In AA, autoimmunity develops against the hair follicle, resulting in non-scarring hair loss that can begin as patches, which can coalesce and progress to cover the entire scalp (alopecia totalis, AT) or eventually the entire body (alopecia universalis, AU) (FIG. 1 ). AA was first described by Cornelius Celsus in 30 A.D., using the term “ophiasis”, which means “snake”, due to the sinuous path of hair loss as it spread slowly across the scalp. Hippocrates first used the Greek word ‘alopekia’ (fox mange), the modern day term “alopecia areata” was first used by Sauvages in his Nosologica Medica, published in 1760 in Lyons, France.

Curiously, AA preferentially affects pigmented hair follicles in the anagen (growth) phase of the hair cycle, and when the hair regrows in patches of AA, it frequently grows back white or colorless. The phenomenon of ‘sudden whitening of the hair’ is therefore ascribed to AA with an acute onset, and has been documented throughout history as having affected several prominent individuals at times of profound grief, stress or fear (2). Examples include Shahjahan, who upon the death of his wife in 1631 experienced acute whitening of his hair, and in his grief built the Taj Mahal in her honor. Sir Thomas More, author of Utopia, who on the eve of his execution in 1535 was said to have become ‘white in both beard and hair’. The sudden whitening of the hair is believed to result from an acute attack upon the pigmented hair follicles, leaving behind the white hairs unscathed.

Several clinical aspects of AA remain unexplained but can hold important clues toward understanding pathogenesis. AA attacks hairs only around the base of the hair follicles, which are surrounded by dense clusters of lymphocytes, resulting in the pathognomic ‘swarm of bees’ appearance on histology. Based on these observations, it is postulated that a signal(s) in the pigmented, anagen hair follicle is emitted which invokes an acute or chronic immune response against the lower end of the hair follicle, leading to hair cycle perturbation, acute hair shedding, hair shaft anomalies and hair breakage. Despite these dramatic perturbations in the hair follicle, there is no permanent organ destruction and the possibility of hair regrowth remains if immune privilege can be restored.

Throughout history, AA has been considered at times to be a neurological disease brought on by stress or anxiety, or as a result of an infectious agent, or even hormonal dysfunction. The concept of a genetically-determined autoimmune mechanism as the basis for AA emerged during the 20^(th) century from multiple lines of evidence. AA hair follicles exhibit an immune infiltrate with activated Th, Tc and NK cells (3, 4) and there is a shift from a suppressive (Th2) to an autoimmune (Th1) cytokine response. The humanized model of AA, which involves transfer of AA patient scalp onto immune-deficient SCID mice illustrates the autoimmune nature of the disease, since transfer of donor T-cells causes hair loss only when co-cultured with hair follicle or human melanoma homogenate (5, 6). Regulatory T cells which serve to maintain immune tolerance are observed in lower numbers in AA tissue (7), and transfer of these cells to C3H/HeJ mice leads to resistance to AA (8). Although AA has long been considered exclusively as a T-cell mediated disease, in recent years, an additional mechanism of disease has been postulated. The hair follicle is defined as one of a select few immune privileged sites in the body, characterized by the presence of extracellular matrix barriers to impede immune cell trafficking, lack of antigen presenting cells, and inhibition of NK cell activity via the local production of immunosuppressive factors and reduced levels of MHC class I expression (9). Thus, the notion of a ‘collapse of immune privilege’ has also been invoked as part of the mechanism by which AA can arise. Support for a genetic basis for AA comes from multiple lines of evidence, including the observed heritability in first degree relatives (10, 11), twin studies (12), and most recently, from the results of our family-based linkage studies (13).

Treatment of Hair Loss Disorders

This invention provides for the discovery that a known therapeutic, for example an inhibitor of a protein tyrosine kinase (PTK) involved in cytokine signaling, such as JAK/STAT proteins Jak 1, Jak2, Jak3 Stat 1 or Stat 2 (e.g., INCB 018424, tofacitinib (CP690550), Janex 1 (WHI-P131), PF-956980, WHI-P154, VX-509, JAK3 Inhibitor IV, NSC114792, or R348), can be used for the treatment of hair loss disorders. Non-limiting examples of hair loss disorders include: androgenetic alopecia, Alopecia areata, telogen effluvium, alopecia areata, alopecia totalis, and alopecia universalis.

An aspect of the invention encompasses a method of treating a hair-loss disorder in a mammalian subject in need thereof, the method comprising administering to the subject an inhibitor of a protein tyrosine kinase (PTK) involved in cytokine signaling. In one embodiment, the inhibitor is a Jak/Stat inhibitor. In a further embodiment, the inhibitor is INCB 018424. In some embodiments, the Jak3 inhibitor is an antibody that specifically binds to a Jak3 protein or a fragment thereof; an antisense RNA or antisense DNA that decreases expression of the gene that encodes the Jak3 protein; an antisense RNA or antisense DNA that decreases expression of the Jak3 protein; a siRNA that specifically targets the Jak3 gene; a small molecule; or a combination thereof. In one embodiment, the inhibitor is a Jak3 inhibitor. In a further embodiment, the inhibitor is tofacitinib (CP690550). In a further embodiment, the small molecule is Janex 1 (WHI-P131), PF-956980, WHI-P154, VX-509, JAK3 Inhibitor IV, NSC114792, or R348. In another embodiment, the hair-loss disorder comprises androgenetic alopecia, telogen effluvium, alopecia areata, telogen effluvium, tinea capitis, alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex, or alopecia universalis

An aspect of the invention provides for a method for inducing hair growth in a subject where the method comprises administering to the subject an effective amount of an inhibitor of a protein tyrosine kinase (PTK) involved in cytokine signaling. In one embodiment, the inhibitor is a Jak/Stat inhibitor. In other embodiments, the inhibitor is INCB 018424. In some embodiments, the Jak3 inhibitor is an antibody that specifically binds to a Jak3 protein or a fragment thereof; an antisense RNA or antisense DNA that decreases expression of the gene that encodes the Jak3 protein; an antisense RNA or antisense DNA that decreases expression of the Jak3 protein; a siRNA that specifically targets the Jak3 gene; a small molecule; or a combination thereof. In one embodiment, the inhibitor is a Jak3 inhibitor. In a further embodiment, the inhibitor is tofacitinib (CP690550). In a further embodiment, the small molecule is Janex 1 (WHI-P131), PF-956980, WHI-P154, VX-509, JAK3 Inhibitor IV, NSC114792, or R348. In some embodiments, the subject is afflicted with a hair-loss disorder. In other embodiments, the hair-loss disorder comprises androgenetic alopecia, telogen effluvium, alopecia areata, telogen effluvium, tinea capitis, alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex, or alopecia universalis. In some embodiments, the modulating compound can also inhibit hair growth, thus it can be used for treatment of hair growth disorders, such as hypertrichosis.

An aspect of the invention encompasses a method of treating a hair-loss disorder in a mammalian subject in need thereof, the method comprising administering to the subject a Jak1 or Jak3 inhibitor. In one embodiment, the inhibitor is an antibody or antibody fragment that is directed to SEQ ID NO: 1 (Jak1), SEQ ID NO: 3 (Jak2), or SEQ ID NO: 109 (Jak3). In another embodiment, the hair-loss disorder comprises androgenetic alopecia, telogen effluvium, alopecia areata, telogen effluvium, tinea capitis, alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex, or alopecia universalis.

An aspect of the invention provides for a method for inducing hair growth in a subject where the method comprises administering to the subject an effective amount of a Jak1, Jak2, or Jak3 inhibitor, thereby controlling hair growth in the subject. In one embodiment, the inhibitor comprises an antibody that specifically binds to a protein comprising SEQ ID NO: 1 (Jak1), SEQ ID NO: 3 (Jak2), or SEQ ID NO: 109 (Jak3). In some embodiments, the subject is afflicted with a hair-loss disorder. In other embodiments, the hair-loss disorder comprises androgenetic alopecia, telogen effluvium, alopecia areata, telogen effluvium, tinea capitis, alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex, or alopecia universalis. In some embodiments, the modulating compound can also inhibit hair growth, thus it can be used for treatment of hair growth disorders, such as hypertrichosis.

An aspect of the invention encompasses a method of treating a hair-loss disorder in a mammalian subject in need thereof, the method comprising administering to the subject a Stat 1 inhibitor. In one embodiment, the inhibitor is an antibody or antibody fragment that is directed to SEQ ID NO: 5. In another embodiment, the hair-loss disorder comprises androgenetic alopecia, telogen effluvium, alopecia areata, telogen effluvium, tinea capitis, alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex, or alopecia universalis

An aspect of the invention provides for a method for inducing hair growth in a subject where the method comprises administering to the subject an effective amount of a Stat 1 inhibitor, thereby controlling hair growth in the subject. In one embodiment, the inhibitor comprises an antibody that specifically binds to a protein comprising SEQ ID NO: 5. In some embodiments, the subject is afflicted with a hair-loss disorder. In other embodiments, the hair-loss disorder comprises androgenetic alopecia, telogen effluvium, alopecia areata, telogen effluvium, tinea capitis, alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex, or alopecia universalis. In some embodiments, the modulating compound can also inhibit hair growth, thus it can be used for treatment of hair growth disorders, such as hypertrichosis.

An aspect of the invention encompasses a method of treating a hair-loss disorder in a mammalian subject in need thereof, the method comprising administering to the subject a Stat 2 inhibitor. In one embodiment, the inhibitor is an antibody or antibody fragment that is directed to SEQ ID NO: 7. In another embodiment, the hair-loss disorder comprises androgenetic alopecia, telogen effluvium, alopecia areata, telogen effluvium, tinea capitis, alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex, or alopecia universalis

An aspect of the invention provides for a method for inducing hair growth in a subject where the method comprises administering to the subject an effective amount of a Stat 2 inhibitor, thereby controlling hair growth in the subject. In one embodiment, the inhibitor comprises an antibody that specifically binds to a protein comprising SEQ ID NO: 7. In some embodiments, the subject is afflicted with a hair-loss disorder. In other embodiments, the hair-loss disorder comprises androgenetic alopecia, telogen effluvium, alopecia areata, telogen effluvium, tinea capitis, alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex, or alopecia universalis. In some embodiments, the modulating compound can also inhibit hair growth, thus it can be used for treatment of hair growth disorders, such as hypertrichosis.

This invention provides for the discovery that a number human genes have been identified as a cohort of genes involved in telogen-to-anagen transition of the hair cycle (e.g, Telogen-to-Anagen Hair Cycle (TAHC) gene). These genes were identified as being upregulated in the telogen phase of the hair cycle, and can be correlated with the presence of a hair loss disorder in a subject. These genes, now that they have been identified, can be used for a variety of useful methods; for example, they can be used to determine whether a subject has susceptibility to a hair-loss disorder, such as Alopecia Areata (AA). The genes identified as part of this telogen-to-anagen transition hair cycle cohort or group (i.e., “TAHC genes”) include CSF1R (Gene ID Accession No. 1436), FCER2 (Gene ID Accession No. 2208), IFNGR1 (Gene ID Accession No. 3459), 1L20 (Gene ID Accession No. 50604), OAS1 (Gene ID Accession No. 4938), PTPRC (Gene ID Accession No. 5788), CEBPD (Gene ID Accession No. 1052), CRP (Gene ID Accession No. 1401), IL2RA (Gene ID Accession No. 3559), IL4 (Gene ID Accession No. 3565), IL6ST (Gene ID Accession No. 3572), INSR (Gene ID Accession No. 3643), JAK3 (Gene ID Accession No. 3718), NR3C1 (Gene ID Accession No. 2908), OSM (Gene ID Accession No. 5008), PTPN11 (Gene ID Accession No. 5781), SOCS3 (Gene ID Accession No. 9021), STAT5A (Gene ID Accession No. 6776), STAT5B (Gene ID Accession No. 6777), CCND1 (Gene ID Accession No. 595), F2 (Gene ID Accession No. 2147), LRG1 (Gene ID Accession No. 116844), PRLR (Gene ID Accession No. 5618), MPL (Gene ID Accession No. 4352), and JUNB (Gene ID Accession No. 3726).

In one embodiment, the invention encompasses a method for detecting the presence of or a predisposition to a hair-loss disorder in a human subject where the method comprises obtaining a biological sample from a human subject; and detecting whether or not there is an alteration in the level of expression of an mRNA or a protein encoded by a TAHC gene in the subject as compared to the level of expression in a subject not afflicted with a hair-loss disorder. In on embodiment, the detecting comprises determining whether mRNA expression or protein expression of the TAHC gene is increased or decreased as compared to expression in a normal sample. In another embodiment, the detecting comprises determining in the sample whether expression of at least 2 TAHC proteins, at least 3 TAHC proteins, at least 4 TAHC proteins, at least 5 TAHC proteins, at least 6 TAHC proteins, at least 6 TAHC proteins, at least 7 TAHC proteins, or at least 8 TAHC proteins is increased or decreased as compared to expression in a normal sample. In some embodiments, the detecting comprises determining in the sample whether expression of at least 2 TAHC mRNAs, at least 3 TAHC mRNAs, at least 4 TAHC mRNAs, at least 5 TAHC mRNAs, at least 6 TAHC mRNAs, at least 6 TAHC mRNAs, at least 7 TAHC mRNAs, or at least 8 TAHC mRNAs is increased or decreased as compared to expression in a normal sample. In one embodiment, an increase in the expression of at least 2 TAHC genes, at least 3 TAHC genes, at least 4 TAHC genes, at least 5 TAHC genes, at least 6 TAHC genes, at least 7 TAHC genes, or at least 8 TAHC genes indicates a predisposition to or presence of a hair-loss disorder in the subject. In another embodiment, a decrease in the expression of at least 2 TAHC genes, at least 3 TAHC genes, at least 4 TAHC genes, at least 5 TAHC genes, at least 6 TAHC genes, at least 7 TAHC genes, or at least 8 TAHC genes indicates a predisposition to or presence of a hair-loss disorder in the subject. In one embodiment, the mRNA expression or protein expression level in the subject is about 5-fold increased, about 10-fold increased, about 15-fold increased, about 20-fold increased, about 25-fold increased, about 30-fold increased, about 35-fold increased, about 40-fold increased, about 45-fold increased, about 50-fold increased, about 55-fold increased, about 60-fold increased, about 65-fold increased, about 70-fold increased, about 75-fold increased, about 80-fold increased, about 85-fold increased, about 90-fold increased, about 95-fold increased, or is 100-fold increased, as compared to that in the normal sample. In some embodiments, the the mRNA expression or protein expression level in the subject is at least about 100-fold increased, at least about 200-fold increased, at least about 300-fold increased, at least about 400-fold increased, or is at least about 500-fold increased, as compared to that in the normal sample. In further embodiments, the mRNA expression or protein expression level of the TAHC gene in the subject is about 5-fold to about 70-fold increased, as compared to that in the normal sample. In other embodiments, the mRNA or protein expression level of the TAHC gene in the subject is about 5-fold to about 90-fold increased, as compared to that in the normal sample. In one embodiment, the mRNA expression or protein expression level in the subject is about 5-fold decreased, about 10-fold decreased, about 15-fold decreased, about 20-fold decreased, about 25-fold decreased, about 30-fold decreased, about 35-fold decreased, about 40-fold decreased, about 45-fold decreased, about 50-fold decreased, about 55-fold decreased, about 60-fold decreased, about 65-fold decreased, about 70-fold decreased, about 75-fold decreased, about 80-fold decreased, about 85-fold decreased, about 90-fold decreased, about 95-fold decreased, or is 100-fold decreased, as compared to that in the normal sample. In some embodiments, the mRNA expression or protein expression level in the subject is at least about 100-fold decreased, as compared to that in the normal sample. In some embodiments, the mRNA or protein expression level of the TAHC gene in the subject is about 5-fold to about 70-fold decreased, as compared to that in the normal sample. In yet other embodiments, the mRNA or protein expression level of the TAHC gene in the subject is about 5-fold to about 90-fold decreased, as compared to that in the normal sample. In further embodiments, the detecting comprises gene sequencing, selective hybridization, selective amplification, gene expression analysis, or a combination thereof. In another embodiment, the hair-loss disorder comprises androgenetic alopecia, alopecia areata, telogen effluvium, alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex, or alopecia universalis. In one embodiment, the TAHC gene is CSF1R, FCER2, IFNGR1, IL20, OAS1, PTPRC, CEBPD, CRP, IL2RA, IL4, IL6ST, INSR, JAK3, NR3C1, OSM, PTPN11, SOCS3, STAT5A, STAT5B, CCND1, F2, LRG1, PRLR, MPL, or JUNB. In another embodiment, the TAHC gene is CRP.

Diagnosis

The invention provides methods to diagnose whether or not a subject is susceptible to or has a hair loss disorder. The diagnostic methods, in one embodiment, are based on monitoring the expression of TAHC genes, such as CSF1R, FCER2, IFNGR1, IL20, OAS1, PTPRC, CEBPD, CRP, IL2RA, IL4, IL6ST, INSR, JAK3, NR3C1, OSM, PTPN11, SOCS3, STAT5A, STAT5B, CCND1, F2, LRG1, PRLR, MPL, or JUNB, in a subject, for example whether they are increased or decreased as compared to a normal sample. As used herein, the term “diagnosis” includes the detection, typing, monitoring, dosing, comparison, at various stages, including early, pre-symptomatic stages, and late stages, in adults and children. Diagnosis can include the assessment of a predisposition or risk of development, the prognosis, or the characterization of a subject to define most appropriate treatment (pharmacogenetics).

The invention provides diagnostic methods to determine whether an individual is at risk of developing a hair-loss disorder, or suffers from a hair-loss disorder, wherein the disease results from an alteration in the expression of TAHC genes. In one embodiment, a method of detecting the presence of or a predisposition to a hair-loss disorder in a subject is provided. The subject can be a human or a child thereof. The method can comprise detecting in a sample from the subject whether or not there is an alteration in the level of expression of a protein encoded by a TAHC gene in the subject as compared to the level of expression in a subject not afflicted with a hair-loss disorder. In one embodiment, the detecting can comprise determining whether mRNA expression of the TAHC is increased or decreased. For example, in a microarray assay, one can look for differential expression of a TAHC gene. Any expression of a TAHC gene that is either 2× higher or 2× lower than TAHC expression observed for a subject not afflicted with a hair-loss disorder (as indicated by a fluorescent read-out) is deemed not normal, and worthy of further investigation. The detecting can also comprise determining in the sample whether expression of at least 2 TAHC proteins, at least 3 TAHC proteins, at least 4 TAHC proteins, at least 5 TAHC proteins, at least 6 TAHC proteins, at least 6 TAHC proteins, at least 7 TAHC proteins, or at least 8 TAHC proteins is increased or decreased. The presence of such an alteration is indicative of the presence or predisposition to a hair-loss disorder.

The presence of an alteration in a TAHC gene in the sample is detected through the genotyping of a sample, for example via gene sequencing, selective hybridization, amplification, gene expression analysis, or a combination thereof. In one embodiment, the sample can comprise blood, serum, sputum, lacrimal secretions, semen, vaginal secretions, fetal tissue, skin tissue, epithelial tissue, muscle tissue, amniotic fluid, or a combination thereof.

The invention provides for a diagnostic kit used to determine whether a sample from a subject exhibits increased expression of at least 2 or more TAHC genes. In one embodiment, the kit comprising a nucleic acid primer that specifically hybridizes to one or more TAHC genes. The invention also provides for a diagnostic kit used to determine whether a sample from a subject exhibits a predisposition to a hair-loss disorder in a human subject. In further embodiments, the TAHC gene is CSF1R, FCER2, IFNGR1, IL20, OAS1, PTPRC, CEBPD, CRP, IL2RA, IL4, IL6ST, INSR, JAK3, NR3C1, OSM, PTPN11, SOCS3, STAT5A, STAT5B, CCND1, F2, LRG1, PRLR, MPL, or JUNB. In another embodiment, the hair-loss disorder comprises androgenetic alopecia, alopecia areata, telogen effluvium, alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex, or alopecia universalis.

DNA and AminoAcid Manipulation Methods and Purification Thereof

The present invention utilizes conventional molecular biology, microbiology, and recombinant DNA techniques available to one of ordinary skill in the art. Such techniques are well known to the skilled worker and are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, “Molecular Cloning: A Laboratory Manual” (1982): “DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover, ed., 1985); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins, eds., 1985); “Transcription and Translation” (B. D. Hames & S. J. Higgins, eds., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1986); “Immobilized Cells and Enzymes” (TRL Press, 1986): B. Perbal, “A Practical Guide to Molecular Cloning” (1984), and Sambrook, et al., “Molecular Cloning: a Laboratory Manual” (1989).

One skilled in the art can obtain a protein in several ways, which include, but are not limited to, isolating the protein via biochemical means or expressing a nucleotide sequence encoding the protein of interest by genetic engineering methods.

A protein is encoded by a nucleic acid (including, for example, genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA). For example, it can be encoded by a recombinant nucleic acid of a gene. The proteins of the invention can be obtained from various sources and can be produced according to various techniques known in the art. For example, a nucleic acid that encodes a protein can be obtained by screening DNA libraries, or by amplification from a natural source. A protein can be a fragment or portion thereof. The nucleic acids encoding a protein can be produced via recombinant DNA technology and such recombinant nucleic acids can be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof. For example, a Jak 1 protein is the polypeptide encoded by the nucleic acid having the nucleotide sequence shown in SEQ ID NO: 2. An example of a Jak 1 polypeptide has the amino acid sequence shown in SEQ ID NO: 1. A Jak 2 protein is the polypeptide encoded by the nucleic acid having the nucleotide sequence shown in SEQ ID NO: 4. An example of a Jak 2 polypeptide has the amino acid sequence shown in SEQ ID NO: 3. A Jak 3 protein is the polypeptide encoded by the nucleic acid having the nucleotide sequence shown in SEQ ID NO: 110. An example of a Jak 3 polypeptide has the amino acid sequence shown in SEQ ID NO: 109. For example, a Stat 1 protein is the polypeptide encoded by the nucleic acid having the nucleotide sequence shown in SEQ ID NO: 6. An example of a Stat 1 polypeptide has the amino acid sequence shown in SEQ ID NO: 5. A Stat 2 protein is the polypeptide encoded by the nucleic acid having the nucleotide sequence shown in SEQ ID NO: 8. An example of a Stat 2 polypeptide has the amino acid sequence shown in SEQ ID NO: 7.

The polypeptide sequence of human Jak 1 is depicted in SEQ ID NO: 1. The nucleotide sequence of human Jak 1 is shown in SEQ ID NO: 2. Sequence information related to Jak 1 is accessible in public databases by GenBank Accession numbers NP_002218 (for protein) and NM_002227.2 (for nucleic acid).

SEQ ID NO: 1 is the human wild type amino acid sequence corresponding to Jak1 (residues 1-1154): 1 MQYLNIKEDC NAMAFCAKMR SSKKTEVNLE APEPGVEVIF YLSDREPLRL GSGEYTAEEL 61 CIRAAQACRI SPLCHNLFAL YDENTKLWYA PNRTITVDDK MSLRLHYRMR FYFTNWHGTN 121 DNEQSVWRHS PKKQKNGYEK KKIPDATPLL DASSLEYLFA QGQYDLVKCL APIRDPKTEQ 181 DGHDIENECL GMAVLAISHY AMMKKMQLPE LPKDISYKRY IPETLNKSIR QRNLLTRMRI 241 NNVFKDFLKE FNNKTICDSS VSTHDLKVKY LATLETLTKH YGAEIFETSM LLISSENEMN 301 WFHSNDGGNV LYYEVMVTGN LGIQWRHKPN VVSVEKEKNK LKRKKLENKH KKDEEKNKIR 361 EEWNNFSYFP EITHIVIKES VVSINKQDNK KMELKLSSHE EALSFVSLVD GYFRLTADAH 421 HYLCTDVAPP LIVHNIQNGC HGPICTEYAI NKLRQEGSEE GMYVLRWSCT DFDNILMTVT 481 CFEKSEQVQG AQKQFKNFQI EVQKGRYSLH GSDRSFPSLG DLMSHLKKQI LRTDNISFML 541 KRCCQPKPRE ISNLLVATKK AQEWQPVYPM SQLSFDRILK KDLVQGEHLG RGTRTHIYSG 601 TLMDYKDDEG TSEEKKIKVI LKVLDPSHRD ISLAFFEAAS MMRQVSHKHI VYLYGVCVRD 661 VENIMVEEFV EGGPLDLFMH RKSDVLTTPW KFKVAKQLAS ALSYLEDKDL VHGNVCTKNL 721 LLAREGIDSE CGPFIKLSDP GIPITVLSRQ ECIERIPWIA PECVEDSKNL SVAADKWSFG 781 TTLWEICYNG EIPLKDKTLI EKERFYESRC RPVTPSCKEL ADLMTRCMNY DPNQRPFFRA 841 IMRDINKLEE QNPDIVSEKK PATEVDPTHF EKRFLKRIRD LGEGHFGKVE LCRYDPEGDN 901 TGEQVAVKSL KPESGGNHIA DLKKEIEILR NLYHENIVKY KGICTEDGGN GIKLIMEFLP 961 SGSLKEYLPK NKNKINLKQQ LKYAVQICKG MDYLGSRQYV HRDLAARNVL VESEHQVKIG 1021 DFGLTKAIET DKEYYTVKDD RDSPVFWYAP ECLMQSKFYI ASDVWSFGVT LHELLTYCDS 1081 DSSPMALFLK MIGPTHGQMT VTRLVNTLKE GKRLPCPPNC PDEVYQLMRK CWEFQPSNRT 1141 SFQNLIEGFE ALLK SEQ ID NO: 2 is the human wild type nucleotide sequence corresponding to Jak1 (nucleotides 1-5053), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame: 1 tgcagacagt gcgggcctgc gcccagtccc ggctgtcctc gccgcgaccc ctcctcagcc 61 ctgggcgcgc gcacgctggg gccccgcggg gctggccgcc tagcgagcct gccggtcgac 121 cccagccagc gcagcgacgg ggcgctgcct ggcccaggcg cacacggaag tgcgcttctc 181 tgaagtagct ttggaaagta gagaagaaaa tccagtttgc ttcttggaga acactggaca 241 gctgaataa a tg cagtatct aaatataaaa gaggactgca atgccatggc tttctgtgct 301 aaaatgagga gctccaagaa gactgaggtg aacctggagg cccctgagcc aggggtggaa 361 gtgatcttct atctgtcgga cagggagccc ctccggctgg gcagtggaga gtacacagca 421 gaggaactgt gcatcagggc tgcacaggca tgccgtatct ctcctctttg tcacaacctc 481 tttgccctgt atgacgagaa caccaagctc tggtatgctc caaatcgcac catcaccgtt 541 gatgacaaga tgtccctccg gctccactac cggatgaggt tctatttcac caattggcat 601 ggaaccaacg acaatgagca gtcagtgtgg cgtcattctc caaagaagca gaaaaatggc 661 tacgagaaaa aaaagattcc agatgcaacc cctctccttg atgccagctc actggagtat 721 ctgtttgctc agggacagta tgatttggtg aaatgcctgg ctcctattcg agaccccaag 781 accgagcagg atggacatga tattgagaac gagtgtctag ggatggctgt cctggccatc 841 tcacactatg ccatgatgaa gaagatgcag ttgccagaac tgcccaagga catcagctac 901 aagcgatata ttccagaaac attgaataag tccatcagac agaggaacct tctcaccagg 961 atgcggataa ataatgtttt caaggatttc ctaaaggaat ttaacaacaa gaccatttgt 1021 gacagcagcg tgtccacgca tgacctgaag gtgaaatact tggctacctt ggaaactttg 1081 acaaaacatt acggtgctga aatatttgag acttccatgt tactgatttc atcagaaaat 1141 gagatgaatt ggtttcattc gaatgacggt ggaaacgttc tctactacga agtgatggtg 1201 actgggaatc ttggaatcca gtggaggcat aaaccaaatg ttgtttctgt tgaaaaggaa 1261 aaaaataaac tgaagcggaa aaaactggaa aataaacaca agaaggatga ggagaaaaac 1321 aagatccggg aagagtggaa caatttttct tacttccctg aaatcactca cattgtaata 1381 aaggagtctg tggtcagcat taacaagcag gacaacaaga aaatggaact gaagctctct 1441 tcccacgagg aggccttgtc ctttgtgtcc ctggtagatg gctacttccg gctcacagca 1501 gatgcccatc attacctctg caccgacgtg gcccccccgt tgatcgtcca caacatacag 1561 aatggctgtc atggtccaat ctgtacagaa tacgccatca ataaattgcg gcaagaagga 1621 agcgaggagg ggatgtacgt gctgaggtgg agctgcaccg actttgacaa catcctcatg 1681 accgtcacct gctttgagaa gtctgagcag gtgcagggtg cccagaagca gttcaagaac 1741 tttcagatcg aggtgcagaa gggccgctac agtctgcacg gttcggaccg cagcttcccc 1801 agcttgggag acctcatgag ccacctcaag aagcagatcc tgcgcacgga taacatcagc 1861 ttcatgctaa aacgctgctg ccagcccaag ccccgagaaa tctccaacct gctggtggct 1921 actaagaaag cccaggagtg gcagcccgtc taccccatga gccagctgag tttcgatcgg 1981 atcctcaaga aggatctggt gcagggcgag caccttggga gaggcacgag aacacacatc 2041 tattctggga ccctgatgga ttacaaggat gacgaaggaa cttctgaaga gaagaagata 2101 aaagtgatcc tcaaagtctt agaccccagc cacagggata tttccctggc cttcttcgag 2161 gcagccagca tgatgagaca ggtctcccac aaacacatcg tgtacctcta tggcgtctgt 2221 gtccgcgacg tggagaatat catggtggaa gagtttgtgg aagggggtcc tctggatctc 2281 ttcatgcacc ggaaaagcga tgtccttacc acaccatgga aattcaaagt tgccaaacag 2341 ctggccagtg ccctgagcta cttggaggat aaagacctgg tccatggaaa tgtgtgtact 2401 aaaaacctcc tcctggcccg tgagggcatc gacagtgagt gtggcccatt catcaagctc 2461 agtgaccccg gcatccccat tacggtgctg tctaggcaag aatgcattga acgaatccca 2521 tggattgctc ctgagtgtgt tgaggactcc aagaacctga gtgtggctgc tgacaagtgg 2581 agctttggaa ccacgctctg ggaaatctgc tacaatggcg agatcccctt gaaagacaag 2641 acgctgattg agaaagagag attctatgaa agccggtgca ggccagtgac accatcatgt 2701 aaggagctgg ctgacctcat gacccgctgc atgaactatg accccaatca gaggcctttc 2761 ttccgagcca tcatgagaga cattaataag cttgaagagc agaatccaga tattgtttca 2821 gaaaaaaaac cagcaactga agtggacccc acacattttg aaaagcgctt cctaaagagg 2881 atccgtgact tgggagaggg ccactttggg aaggttgagc tctgcaggta tgaccccgaa 2941 ggggacaata caggggagca ggtggctgtt aaatctctga agcctgagag tggaggtaac 3001 cacatagctg atctgaaaaa ggaaatcgag atcttaagga acctctatca tgagaacatt 3061 gtgaagtaca aaggaatctg cacagaagac ggaggaaatg gtattaagct catcatggaa 3121 tttctgcctt cgggaagcct taaggaatat cttccaaaga ataagaacaa aataaacctc 3181 aaacagcagc taaaatatgc cgttcagatt tgtaagggga tggactattt gggttctcgg 3241 caatacgttc accgggactt ggcagcaaga aatgtccttg ttgagagtga acaccaagtg 3301 aaaattggag acttcggttt aaccaaagca attgaaaccg ataaggagta ttacaccgtc 3361 aaggatgacc gggacagccc tgtgttttgg tatgctccag aatgtttaat gcaatctaaa 3421 ttttatattg cctctgacgt ctggtctttt ggagtcactc tgcatgagct gctgacttac 3481 tgtgattcag attctagtcc catggctttg ttcctgaaaa tgataggccc aacccatggc 3541 cagatgacag tcacaagact tgtgaatacg ttaaaagaag gaaaacgcct gccgtgccca 3601 cctaactgtc cagatgaggt ttatcaactt atgaggaaat gctgggaatt ccaaccatcc 3661 aatcggacaa gctttcagaa ccttattgaa ggatttgaag cacttttaaa ataagaagca 3721 tgaataacat ttaaattcca cagattatca agtccttctc ctgcaacaaa tgcccaagtc 3781 attttttaaa aatttctaat gaaagaagtt tgtgttctgt ccaaaaagtc actgaactca 3841 tacttcagta catatacatg tataaggcac actgtagtgc ttaatatgtg taaggacttc 3901 ctctttaaat ttggtaccag taacttagtg acacataatg acaaccaaaa tatttgaaag 3961 cacttaagca ctcctccttg tggaaagaat ataccaccat ttcatctggc tagttcacca 4021 tcacaactgc attaccaaaa ggggattttt gaaaacgagg agttgaccaa aataatatct 4081 gaagatgatt gcttttccct gctgccagct gatctgaaat gttttgctgg cacattaatc 4141 atagataaag aaagattgat ggacttagcc ctcaaatttc agtatctata cagtactaga 4201 ccatgcattc ttaaaatatt agataccagg tagtatatat tgtttctgta caaaaatgac 4261 tgtattctct caccagtagg acttaaactt tgtttctcca gtggcttagc tcctgttcct 4321 ttgggtgatc actagcaccc atttttgaga aagctggttc tacatggggg gatagctgtg 4381 gaatagataa tttgctgcat gttaattctc aagaactaag cctgtgccag tgctttccta 4441 agcagtatac ctttaatcag aactcattcc cagaacctgg atgctattac acatgctttt 4501 aagaaacgtc aatgtatatc cttttataac tctaccactt tggggcaagc tattccagca 4561 ctggttttga atgctgtatg caaccagtct gaataccaca tacgctgcac tgttcttaga 4621 gggtttccat acttaccacc gatctacaag ggttgatccc tgtttttacc atcaatcatc 4681 accctgtggt gcaacacttg aaagacccgg ctagaggcac tatggacttc aggatccact 4741 agacagtttt cagtttgctt ggaggtagct gggtaatcaa aaatgtttag tcattgattc 4801 aatgtgaacg attacggtct ttatgaccaa gagtctgaaa atctttttgt tatgctgttt 4861 agtattcgtt tgatattgtt acttttcacc tgttgagccc aaattcagga ttggttcagt 4921 ggcagcaatg aagttgccat ttaaatttgt tcatagccta catcaccaag gtctctgtgt 4981 caaacctgtg gccactctat atgcactttg tttactcttt atacaaataa atatactaaa 5041 gactttacat gca

The polypeptide sequence of human Jak 2 is depicted in SEQ ID NO: 3. The nucleotide sequence of human Jak 2 is shown in SEQ ID NO: 4. Sequence information related to Jak 2 is accessible in public databases by GenBank Accession numbers NP_004963 (for protein) and NM_004972.3 (for nucleic acid).

SEQ ID NO: 3 is the human wild type amino acid sequence corresponding to Jak2 (residues 1-1132): 1 MGMACLTMTE MEGTSTSSIY QNGDISGNAN SMKQIDPVLQ VYLYHSLGKS EADYLTFPSG 61 EYVAEEICIA ASKACGITPV YHNMFALMSE TERIWYPPNH VFHIDESTRH NVLYRIRFYF 121 PRWYCSGSNR AYRHGISRGA EAPLLDDFVM SYLFAQWRHD FVHGWIKVPV THETQEECLG 181 MAVLDMMRIA KENDQTPLAI YNSISYKTFL PKCIRAKIQD YHILTRKRIR YRFRRFIQQF 241 SQCKATARNL KLKYLINLET LQSAFYTEKF EVKEPGSGPS GEEIFATIII TGNGGIQWSR 301 GKHKESETLT EQDLQLYCDF PNIIDVSIKQ ANQEGSNESR VVTIHKQDGK NLEIELSSLR 361 EALSFVSLID GYYRLTADAH HYLCKEVAPP AVLENIQSNC HGPISMDFAI SKLKKAGNQT 421 GLYVLRCSPK DFNKYFLTFA VERENVIEYK HCLITKNENE EYNLSGTKKN FSSLKDLLNC 481 YQMETVRSDN IIFQFTKCCP PKPKDKSNLL VFRTNGVSDV PTSPTLQRPT HMNQMVFHKI 541 RNEDLIFNES LGQGTFTKIF KGVRREVGDY GQLHETEVLL KVLDKAHRNY SESFFEAASM 601 MSKLSHKHLV LNYGVCVCGD ENILVQEFVK FGSLDTYLKK NKNCINILWK LEVAKQLAWA 661 MHFLEENTLI HGNVCAKNIL LIREEDRKTG NPPFIKLSDP GISITVLPKD ILQERIPWVP 721 PECIENPKNL NLATDKWSFG TTLWEICSGG DKPLSALDSQ RKLQFYEDRH QLPAPKWAEL 781 ANLINNCMDY EPDFRPSFRA IIRDLNSLFT PDYELLTEND MLPNMRIGAL GFSGAFEDRD 841 PTQFEERHLK FLQQLGKGNF GSVEMCRYDP LQDNTGEVVA VKKLQHSTEE HLRDFEREIE 901 ILKSLQHDNI VKYKGVCYSA GRRNLKLIME YLPYGSLRDY LQKHKERIDH IKLLQYTSQI 961 CKGMEYLGTK RYIHRDLATR NILVENENRV KIGDFGLTKV LPQDKEYYKV KEPGESPIFW 1021 YAPESLTESK FSVASDVWSF GVVLYELFTY IEKSKSPPAE FMRMIGNDKQ GQMIVFHLIE 1081 LLKNNGRLPR PDGCPDEIYM IMTECWNNNV NQRPSFRDLA LRVDQIRDNM AG SEQ ID NO: 4 is the human wild type nucleotide sequence corresponding to Jak2 (nucleotides 1-5285), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame: 1 ctgcaggaag gagagaggaa gaggagcaga agggggcagc agcggacgcc gctaacggcc 61 tccctcggcg ctgacaggct gggccggcgc ccggctcgct tgggtgttcg cgtcgccact 121 tcggcttctc ggccggtcgg gcccctcggc ccgggcttgc ggcgcgcgtc ggggctgagg 181 gctgctgcgg cgcagggaga ggcctggtcc tcgctgccga gggatgtgag tgggagctga 241 gcccacactg gagggccccc gagggcccag cctggaggtc gttcagagcc gtgcccgtcc 301 cggggcttcg cagaccttga cccgccgggt aggagccgcc cctgcgggct cgagggcgcg 361 ctctggtcgc ccgatctgtg tagccggttt cagaagcagg caacaggaac aagatgtgaa 421 ctgtttctct tctgcagaaa aagaggctct tcctcctcct cccgcgacgg caaatgttct 481 gaaaaagact ctgc atg gga atggcctgcc ttacgatgac agaaatggag ggaacatcca 541 cctcttctat atatcagaat ggtgatattt ctggaaatgc caattctatg aagcaaatag 601 atccagttct tcaggtgtat ctttaccatt cccttgggaa atctgaggca gattatctga 661 cctttccatc tggggagtat gttgcagaag aaatctgtat tgctgcttct aaagcttgtg 721 gtatcacacc tgtgtatcat aatatgtttg ctttaatgag tgaaacagaa aggatctggt 781 atccacccaa ccatgtcttc catatagatg agtcaaccag gcataatgta ctctacagaa 841 taagatttta ctttcctcgt tggtattgca gtggcagcaa cagagcctat cggcatggaa 901 tatctcgagg tgctgaagct cctcttcttg atgactttgt catgtcttac ctctttgctc 961 agtggcggca tgattttgtg cacggatgga taaaagtacc tgtgactcat gaaacacagg 1021 aagaatgtct tgggatggca gtgttagata tgatgagaat agccaaagaa aacgatcaaa 1081 ccccactggc catctataac tctatcagct acaagacatt cttaccaaaa tgtattcgag 1141 caaagatcca agactatcat attttgacaa ggaagcgaat aaggtacaga tttcgcagat 1201 ttattcagca attcagccaa tgcaaagcca ctgccagaaa cttgaaactt aagtatctta 1261 taaatctgga aactctgcag tctgccttct acacagagaa atttgaagta aaagaacctg 1321 gaagtggtcc ttcaggtgag gagatttttg caaccattat aataactgga aacggtggaa 1381 ttcagtggtc aagagggaaa cataaagaaa gtgagacact gacagaacag gatttacagt 1441 tatattgcga ttttcctaat attattgatg tcagtattaa gcaagcaaac caagagggtt 1501 caaatgaaag ccgagttgta actatccata agcaagatgg taaaaatctg gaaattgaac 1561 ttagctcatt aagggaagct ttgtctttcg tgtcattaat tgatggatat tatagattaa 1621 ctgcagatgc acatcattac ctctgtaaag aagtagcacc tccagccgtg cttgaaaata 1681 tacaaagcaa ctgtcatggc ccaatttcga tggattttgc cattagtaaa ctgaagaaag 1741 caggtaatca gactggactg tatgtacttc gatgcagtcc taaggacttt aataaatatt 1801 ttttgacttt tgctgtcgag cgagaaaatg tcattgaata taaacactgt ttgattacaa 1861 aaaatgagaa tgaagagtac aacctcagtg ggacaaagaa gaacttcagc agtcttaaag 1921 atcttttgaa ttgttaccag atggaaactg ttcgctcaga caatataatt ttccagttta 1981 ctaaatgctg tcccccaaag ccaaaagata aatcaaacct tctagtcttc agaacgaatg 2041 gtgtttctga tgtaccaacc tcaccaacat tacagaggcc tactcatatg aaccaaatgg 2101 tgtttcacaa aatcagaaat gaagatttga tatttaatga aagccttggc caaggcactt 2161 ttacaaagat ttttaaaggc gtacgaagag aagtaggaga ctacggtcaa ctgcatgaaa 2221 cagaagttct tttaaaagtt ctggataaag cacacagaaa ctattcagag tctttctttg 2281 aagcagcaag tatgatgagc aagctttctc acaagcattt ggttttaaat tatggagtat 2341 gtgtctgtgg agacgagaat attctggttc aggagtttgt aaaatttgga tcactagata 2401 catatctgaa aaagaataaa aattgtataa atatattatg gaaacttgaa gttgctaaac 2461 agttggcatg ggccatgcat tttctagaag aaaacaccct tattcatggg aatgtatgtg 2521 ccaaaaatat tctgcttatc agagaagaag acaggaagac aggaaatcct cctttcatca 2581 aacttagtga tcctggcatt agtattacag ttttgccaaa ggacattctt caggagagaa 2641 taccatgggt accacctgaa tgcattgaaa atcctaaaaa tttaaatttg gcaacagaca 2701 aatggagttt tggtaccact ttgtgggaaa tctgcagtgg aggagataaa cctctaagtg 2761 ctctggattc tcaaagaaag ctacaatttt atgaagatag gcatcagctt cctgcaccaa 2821 agtgggcaga attagcaaac cttataaata attgtatgga ttatgaacca gatttcaggc 2881 cttctttcag agccatcata cgagatctta acagtttgtt tactccagat tatgaactat 2941 taacagaaaa tgacatgtta ccaaatatga ggataggtgc cctggggttt tctggtgcct 3001 ttgaagaccg ggatcctaca cagtttgaag agagacattt gaaatttcta cagcaacttg 3061 gcaagggtaa ttttgggagt gtggagatgt gccggtatga ccctctacag gacaacactg 3121 gggaggtggt cgctgtaaaa aagcttcagc atagtactga agagcaccta agagactttg 3181 aaagggaaat tgaaatcctg aaatccctac agcatgacaa cattgtaaag tacaagggag 3241 tgtgctacag tgctggtcgg cgtaatctaa aattaattat ggaatattta ccatatggaa 3301 gtttacgaga ctatcttcaa aaacataaag aacggataga tcacataaaa cttctgcagt 3361 acacatctca gatatgcaag ggtatggagt atcttggtac aaaaaggtat atccacaggg 3421 atctggcaac gagaaatata ttggtggaga acgagaacag agttaaaatt ggagattttg 3481 ggttaaccaa agtcttgcca caagacaaag aatactataa agtaaaagaa cctggtgaaa 3541 gtcccatatt ctggtatgct ccagaatcac tgacagagag caagttttct gtggcctcag 3601 atgtttggag ctttggagtg gttctgtatg aacttttcac atacattgag aagagtaaaa 3661 gtccaccagc ggaatttatg cgtatgattg gcaatgacaa acaaggacag atgatcgtgt 3721 tccatttgat agaacttttg aagaataatg gaagattacc aagaccagat ggatgcccag 3781 atgagatcta tatgatcatg acagaatgct ggaacaataa tgtaaatcaa cgcccctcct 3841 ttagggatct agctcttcga gtggatcaaa taagggataa catggctgga tgaaagaaat 3901 gaccttcatt ctgagaccaa agtagattta cagaacaaag ttttatattt cacattgctg 3961 tggactatta ttacatatat cattattata taaatcatga tgctagccag caaagatgtg 4021 aaaatatctg ctcaaaactt tcaaagttta gtaagttttt cttcatgagg ccaccagtaa 4081 aagacattaa tgagaattcc ttagcaagga ttttgtaaga agtttcttaa acattgtcag 4141 ttaacatcac tcttgtctgg caaaagaaaa aaaatagact ttttcaactc agctttttga 4201 gacctgaaaa aattattatg taaattttgc aatgttaaag atgcacagaa tatgtatgta 4261 tagtttttac cacagtggat gtataatacc ttggcatctt gtgtgatgtt ttacacacat 4321 gagggctggt gttcattaat actgttttct aatttttcca tagttaatct ataattaatt 4381 acttcactat acaaacaaat taagatgttc agataattga ataagtacct ttgtgtcctt 4441 gttcatttat atcgctggcc agcattataa gcaggtgtat acttttagct tgtagttcca 4501 tgtactgtaa atatttttca cataaaggga acaaatgtct agttttattt gtataggaaa 4561 tttccctgac cctaaataat acattttgaa atgaaacaag cttacaaaga tataatctat 4621 tttattatgg tttcccttgt atctatttgt ggtgaatgtg ttttttaaat ggaactatct 4681 ccaaattttt ctaagactac tatgaacagt tttcttttaa aattttgaga ttaagaatgc 4741 caggaatatt gtcatccttt gagctgctga ctgccaataa cattcttcga tctctgggat 4801 ttatgctcat gaactaaatt taagcttaag ccataaaata gattagattg ttttttaaaa 4861 atggatagct cattaagaag tgcagcaggt taagaatttt ttcctaaaga ctgtatattt 4921 gaggggtttc agaattttgc attgcagtca tagaagagat ttatttcctt tttagagggg 4981 aaatgaggta aataagtaaa aaagtatgct tgttaatttt attcaagaat gccagtagaa 5041 aattcataac gtgtatcttt aagaaaaatg agcatacatc ttaaatcttt tcaattaagt 5101 ataaggggtt gttcgttgtt gtcatttgtt atagtgctac tccactttag acaccatagc 5161 taaaataaaa tatggtgggt tttgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 5221 tgttatttat acaaaactta aaatacttgc tgttttgatt aaaaagaaaa tagtttctta 5281 cttta

The polypeptide sequence of human Jak 3 is depicted in SEQ ID NO: 109. The nucleotide sequence of human Jak 3 is shown in SEQ ID NO: 110. Sequence information related to Jak 3 is accessible in public databases by GenBank Accession numbers NP_000206 (for protein) and NM_000215 (for nucleic acid). JAK3 is a downstream signaling partner of the IL-2 receptor common gamma chain, which is shared with the IL-2, -4, -7, -9, -15, and -21 receptors.

SEQ ID NO: 109 is the human wild type amino acid sequence corresponding to Jak3 (residues 1-1124): 1 MAPPSEETPL IPQRSCSLLS TEAGALHVLL PARGPGPPQR LSFSFGDHLA EDLCVQAAKA 61 SGILPVYHSL FALATEDLSC WFPPSHIFSV EDASTQVLLY RIRFYFPNWF GLEKCHRFGL 121 RKDLASAILD LPVLEHLFAQ HRSDLVSGRL PVGLSLKEQG ECLSLAVLDL ARMAREQAQR 181 PGELLKTVSY KACLPPSLRD LIQGLSFVTR RRIRRTVRRA LRRVAACQAD RHSLMAKYIM 241 DLERLDPAGA AETFHVGLPG ALGGHDGLGL LRVAGDGGIA WTQGEQEVLQ PFCDFPEIVD 301 ISIKQAPRVG PAGEHRLVTV TRTDNQILEA EFPGLPEALS FVALVDGYFR LTTDSQHFFC 361 KEVAPPRLLE EVAEQCHGPI TLDFAINKLK TGGSRPGSYV LRRSPQDFDS FLLTVCVQNP 421 LGPDYKGCLI RRSPTGTFLL VGLSRPHSSL RELLATCWDG GLHVDGVAVT LTSCCIPRPK 481 EKSNLIVVQR GHSPPTSSLV QPQSQYQLSQ MTFHKIPADS LEWHENLGHG SFTKIYRGCR 541 HEVVDGEARK TEVLLKVMDA KHKNCMESFL EAASLMSQVS YRHLVLLHGV CMAGDSTMVQ 601 EFVHLGAIDM YLRKRGHLVP ASWKLQVVKQ LAYALNYLED KGLPHGNVSA RKVLLAREGA 661 DGSPPFIKLS DPGVSPAVLS LEMLTDRIPW VAPECLREAQ TLSLEADKWG FGATVWEVFS 721 GVTMPISALD PAKKLQFYED RQQLPAPKWT ELALLIQQCM AYEPVQRPSF RAVIRDLNSL 781 ISSDYELLSD PTPGALAPRD GLWNGAQLYA CQDPTIFEER HLKYISQLGK GNFGSVELCR 841 YDPLGDNTGA LVAVKQLQHS GPDQQRDFQR EIQILKALHS DFIVKYRGVS YGPGRQSLRL 901 VMEYLPSGCL RDFLQRHRAR LDASRLLLYS SQICKGMEYL GSRRCVHRDL AARNILVESE 961 AHVKIADFGL AKLLPLDKDY YVVREPGQSP IFWYAPESLS DNIFSRQSDV WSFGVVLYEL 1021 FTYCDKSCSP SAEFLRMMGC ERDVPALCRL LELLEEGQRL PAPPACPAEV HELMKLCWAP 1081 SPQDRPSFSA LGPQLDMLWS GSRGCETHAF TAHPEGKHHS LSFS SEQ ID NO: 110 is the human wild type nucleotide sequence corresponding to Jak3 (nucleotides 1-5449), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame: 1 cacacaggaa ggagccgagt gggactttcc tctcgctgcc tcccggctct gcccgccctt 61 cgaaagtcca gggtccctgc ccgctaggca agttgcactc  atg gcacctc caagtgaaga 121 gacgcccctg atccctcagc gttcatgcag cctcttgtcc acggaggctg gtgccctgca 181 tgtgctgctg cccgctcggg gccccgggcc cccccagcgc ctatctttct cctttgggga 241 ccacttggct gaggacctgt gcgtgcaggc tgccaaggcc agcggcatcc tgcctgtgta 301 ccactccctc tttgctctgg ccacggagga cctgtcctgc tggttccccc cgagccacat 361 cttctccgtg gaggatgcca gcacccaagt cctgctgtac aggattcgct tttacttccc 421 caattggttt gggctggaga agtgccaccg cttcgggcta cgcaaggatt tggccagtgc 481 tatccttgac ctgccagtcc tggagcacct ctttgcccag caccgcagtg acctggtgag 541 tgggcgcctc cccgtgggcc tcagtctcaa ggagcagggt gagtgtctca gcctggccgt 601 gttggacctg gcccggatgg cgcgagagca ggcccagcgg ccgggagagc tgctgaagac 661 tgtcagctac aaggcctgcc tacccccaag cctgcgcgac ctgatccagg gcctgagctt 721 cgtgacgcgg aggcgtattc ggaggacggt gcgcagagcc ctgcgccgcg tggccgcctg 781 ccaggcagac cggcactcgc tcatggccaa gtacatcatg gacctggagc ggctggatcc 841 agccggggcc gccgagacct tccacgtggg cctccctggg gcccttggtg gccacgacgg 901 gctggggctg ctccgcgtgg ctggtgacgg cggcatcgcc tggacccagg gagaacagga 961 ggtcctccag cccttctgcg actttccaga aatcgtagac attagcatca agcaggcccc 1021 gcgcgttggc ccggccggag agcaccgcct ggtcactgtt accaggacag acaaccagat 1081 tttagaggcc gagttcccag ggctgcccga ggctctgtcg ttcgtggcgc tcgtggacgg 1141 ctacttccgg ctgaccacgg actcccagca cttcttctgc aaggaggtgg caccgccgag 1201 gctgctggag gaagtggccg agcagtgcca cggccccatc actctggact ttgccatcaa 1261 caagctcaag actgggggct cacgtcctgg ctcctatgtt ctccgccgca gcccccagga 1321 ctttgacagc ttcctcctca ctgtctgtgt ccagaacccc cttggtcctg attataaggg 1381 ctgcctcatc cggcgcagcc ccacaggaac cttccttctg gttggcctca gccgacccca 1441 cagcagtctt cgagagctcc tggcaacctg ctgggatggg gggctgcacg tagatggggt 1501 ggcagtgacc ctcacttcct gctgtatccc cagacccaaa gaaaagtcca acctgatcgt 1561 ggtccagaga ggtcacagcc cacccacatc atccttggtt cagccccaat cccaatacca 1621 gctgagtcag atgacatttc acaagatccc tgctgacagc ctggagtggc atgagaacct 1681 gggccatggg tccttcacca agatttaccg gggctgtcgc catgaggtgg tggatgggga 1741 ggcccgaaag acagaggtgc tgctgaaggt catggatgcc aagcacaaga actgcatgga 1801 gtcattcctg gaagcagcga gcttgatgag ccaagtgtcg taccggcatc tcgtgctgct 1861 ccacggcgtg tgcatggctg gagacagcac catggtgcag gaatttgtac acctgggggc 1921 catagacatg tatctgcgaa aacgtggcca cctggtgcca gccagctgga agctgcaggt 1981 ggtcaaacag ctggcctacg ccctcaacta tctggaggac aaaggcctgc cccatggcaa 2041 tgtctctgcc cggaaggtgc tcctggctcg ggagggggct gatgggagcc cgcccttcat 2101 caagctgagt gaccctgggg tcagccccgc tgtgttaagc ctggagatgc tcaccgacag 2161 gatcccctgg gtggcccccg agtgtctccg ggaggcgcag acacttagct tggaagctga 2221 caagtggggc ttcggcgcca cggtctggga agtgtttagt ggcgtcacca tgcccatcag 2281 tgccctggat cctgctaaga aactccaatt ttatgaggac cggcagcagc tgccggcccc 2341 caagtggaca gagctggccc tgctgattca acagtgcatg gcctatgagc cggtccagag 2401 gccctccttc cgagccgtca ttcgtgacct caatagcctc atctcttcag actatgagct 2461 cctctcagac cccacacctg gtgccctggc acctcgtgat gggctgtgga atggtgccca 2521 gctctatgcc tgccaagacc ccacgatctt cgaggagaga cacctcaagt acatctcaca 2581 gctgggcaag ggcaactttg gcagcgtgga gctgtgccgc tatgacccgc taggcgacaa 2641 tacaggtgcc ctggtggccg tgaaacagct gcagcacagc gggccagacc agcagaggga 2701 ctttcagcgg gagattcaga tcctcaaagc actgcacagt gatttcattg tcaagtatcg 2761 tggtgtcagc tatggcccgg gccgccagag cctgcggctg gtcatggagt acctgcccag 2821 cggctgcttg cgcgacttcc tgcagcggca ccgcgcgcgc ctcgatgcca gccgcctcct 2881 tctctattcc tcgcagatct gcaagggcat ggagtacctg ggctcccgcc gctgcgtgca 2941 ccgcgacctg gccgcccgaa acatcctcgt ggagagcgag gcacacgtca agatcgctga 3001 cttcggccta gctaagctgc tgccgcttga caaagactac tacgtggtcc gcgagccagg 3061 ccagagcccc attttctggt atgcccccga atccctctcg gacaacatct tctctcgcca 3121 gtcagacgtc tggagcttcg gggtcgtcct gtacgagctc ttcacctact gcgacaaaag 3181 ctgcagcccc tcggccgagt tcctgcggat gatgggatgt gagcgggatg tccccgccct 3241 ctgccgcctc ttggaactgc tggaggaggg ccagaggctg ccggcgcctc ctgcctgccc 3301 tgctgaggtt cacgagctca tgaagctgtg ctgggcccct agcccacagg accggccatc 3361 attcagcgcc ctgggccccc agctggacat gctgtggagc ggaagccggg ggtgtgagac 3421 tcatgccttc actgctcacc cagagggcaa acaccactcc ctgtcctttt catagctcct 3481 gcccgcagac ctctggatta ggtctctgtt gactggctgt gtgaccttag gcccggagct 3541 gcccctctct gggcctcaga ggccttatga gggtcctcta cttcaggaac acccccatga 3601 cattgcattt gggggggctc ccgtggcctg tagaatagcc tgtggccttt gcaatttgtt 3661 aaggttcaag acagatgggc atatgtgtca gtggggctct ctgagtcctg gcccaaagaa 3721 gcaaggaacc aaatttaaga ctctcgcatc ttcccaaccc cttaagccct ggccccctga 3781 gtttcctttt ctgtctctct ctttttattt tttttatttt tatttttatt tttgagacag 3841 agcctcgctc tgttacccag ggtggagtgc agtggtgcga tctcggctca gtgcaacctc 3901 tgcttcccag gttcaagcga ttctcctgcc tcagcctccc gagtagctgg gattacaggt 3961 gtgcaccacc acacccggct aatttttttt atttttaata gagatgaggt ttcaccatga 4021 tggccaggct gatctcgaac tcctaacctc aagtgatcct cccacctcag cctcccaaag 4081 tgttggaata ataggcatga gccactgcac ccaggctttt ttttttttaa atttattatt 4141 attattttta agagacagga tcttgctacg ttgcccaggc tggtcttgaa ctcctgggct 4201 acagtgatcc tcctgcctta tcctcctaaa tagctgggac tacagcacct agttttgagt 4261 ttcctgtctt atttccaatg gggacattca tgtagctttt tttttttttt tttttttgag 4321 acggagtctc gctctgtcgc ccaggctgga gtacagtggc gcaatctagg ctcactgcaa 4381 gctccgcctc ctgggttcac accattctct cgcctcagcc tcccaagtag ctgggactac 4441 aggcgcccgc caccacaccc ggctaatttt ttgtattttt agtagagacg gggtttcacc 4501 ttgttagcca ggatggtttc catctcctga cctcgtgatc tgcccgtctc ggcctcccaa 4561 agtgctggga ttacaggcat gagccactgc gcccggccct catgtagctt taaatgtatg 4621 atctgacttc tgctccccga tctctgtttc tctggaggaa gccaaggaca agagcagttg 4681 ctgtggctgg gactctgcct tttaggggag cccgtgtatc tctttgggat cctgaaaggg 4741 ggcaggaaag gctggggtcc cagtccaccc taatggtatc tgagtgtcct agggcttcag 4801 ttttcccacc tgtccaatgg gaccctttct gtcctcaccc tacaaggggc acaaagggat 4861 gacaccaaac ctggcaggaa cttttcacgc aatcaaggga aggaaaggca ttcctggcag 4921 agggaacagc atgccaagcg tgagaaggct cagagtaagg aggttaagag cccaagtatt 4981 ggagcctaca gttttgcccc ttccatgcag tgtgacagtg ggcaagttcc tttccctctc 5041 tgggtctcag ttctgtcccc tgcaaaatgg tcagagctta ccccttggct gtgcagggtc 5101 aactttctga ctggtgagag ggattctcat gcaggttaag cttctgctgc tcctcctcac 5161 ctgcaaagct tttctgccac ttttgcctcc ttggaaaact cttatccatc tctcaaaact 5221 ccagctacca catccttgca gccttccctc atataccccc actactactg tagccctgtc 5281 cttccctcca gccccactct ggccctgggg ctggggaagt gtctgtgtcc agctgtctcc 5341 cctgacctca gggttccttg ggggctgggc tgaggcctca gtacagaggg ggctctggaa 5401 atgtttgttg actgaataaa ggaattcagt ggaaaaaaaa aaaaaaaaa

The polypeptide sequence of human Stat 1 is depicted in SEQ ID NO: 5. The nucleotide sequence of human Stat 1 is shown in SEQ ID NO: 6. Sequence information related to Stat 1 is accessible in public databases by GenBank Accession numbers ADA59516 (for protein) and GU211347.1 (for nucleic acid).

SEQ ID NO: 5 is the human wild type amino acid sequence corresponding to Stat1 (residues 1-750): 1 MSQWYELQQL DSKFLEQVHQ LYDDSFPMEI RQYLAQWLEK QDWEHTANDV SFATIRFHDL 61 LSQLDDQYSR FSLENNFLLQ HNIRKSKRNL QDNFQEDPIQ MSMIIYSCLK EERKILENAQ 121 RFNQAQSGNI QSTVMLDKQK ELDSKVRNVK DKVMCIEHEI KSLEDLQDEY DFKCKTLQNR 181 EHETNGVAKS DQKQEQLLLK KMYLMLDNKR KEVVHKIIEL LNVTELTQNA LINDELVEWK 241 RRQQSACIGG PPNACLDQLQ NWFTIVAESL QQVRQQLKKL EELEQKYTYE HDPITKNKQV 301 LWDRTFSLFQ QLIQSSFVVE RQPCMPTHPQ RPLVLKTGVQ FTVKLRLLVK LQELNYNLKV 361 KVLFDKDVNE RNTVKGFRKF NILGTHTKVM NMEESTNGSL AAEFRHLQLK EQKNAGTRTN 421 EGPLIVTEEL HSLSFETQLC QPGLVIDLET TSLPVVVISN VSQLPSGWAS ILWYNMLVAE 481 PRNLSFFLTP PCARWAQLSE VLSWQFSSVT KRGLNVDQLN MLGEKLLGPN ASPDGLIPWT 541 RFCKENINDK NFPFWLWIES ILELIKKHLL PLWNDGCIMG FISKERERAL LKDQQPGTFL 601 LRFSESSREG AITFTWVERS QNGGEPDFHA VEPYTKKELS AVTFPDIIRN YKVMAAENIP 661 ENPLKYLYPN IDKDHAFGKY YSRPKEAPEP MELDGPKGTG YIKTELISVS EVHPSRLQTT 721 DNLLPMSPEE FDEVSRIVGS VEFDSMMNTV SEQ ID NO: 6 is the human wild type nucleotide sequence corresponding to Stat1 (nucleotides 1-2353), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame: 1 gtgctgtgcg tagctgctcc tttggttgaa tccccaggcc cttgttgggg cacaaggtgg 61 cagg atg tct cagtggtacg aacttcagca gcttgactca aaattcctgg agcaggttca 121 ccagctttat gatgacagtt ttcccatgga aatcagacag tacctggcac agtggttaga 181 aaagcaagac tgggagcaca ctgccaatga tgtttcattt gccaccatcc gttttcatga 241 cctcctgtca cagctggatg atcaatatag tcgcttttct ttggagaata acttcttgct 301 acagcataac ataaggaaaa gcaagcgtaa tcttcaggat aattttcagg aagacccaat 361 ccagatgtct atgatcattt acagctgtct gaaggaagaa aggaaaattc tggaaaacgc 421 ccagagattt aatcaggctc agtcggggaa tattcagagc acagtgatgt tagacaaaca 481 gaaagagctt gacagtaaag tcagaaatgt gaaggacaag gttatgtgta tagagcatga 541 aatcaagagc ctggaagatt tacaagatga atatgacttc aaatgcaaaa ccttgcagaa 601 cagagaacac gagaccaatg gtgtggcaaa gagtgatcag aaacaagaac agctgttact 661 caagaagatg tatttaatgc ttgacaataa gagaaaggaa gtagttcaca aaataataga 721 gttgctgaat gtcactgaac ttacccagaa tgccctgatt aatgatgaac tagtggagtg 781 gaagcggaga cagcagagcg cctgtattgg ggggccgccc aatgcttgct tggatcagct 841 gcagaactgg ttcactatag ttgcggagag tctgcagcaa gttcggcagc agcttaaaaa 901 gttggaggaa ttggaacaga aatacaccta cgaacatgac cctatcacaa aaaacaaaca 961 agtgttatgg gaccgcacct tcagtctttt ccagcagctc attcagagct cgtttgtggt 1021 ggaaagacag ccctgcatgc caacgcaccc tcagaggccg ctggtcttga agacaggggt 1081 ccagttcact gtgaagttga gactgttggt gaaattgcaa gagctgaatt ataatttgaa 1141 agtcaaagtc ttatttgata aagatgtgaa tgagagaaat acagtaaaag gatttaggaa 1201 gttcaacatt ttgggcacgc acacaaaagt gatgaacatg gaggagtcca ccaatggcag 1261 tctggcggct gaatttcggc acctgcaatt gaaagaacag aaaaatgctg gcaccagaac 1321 gaatgagggt cctctcatcg ttactgaaga gcttcactcc cttagttttg aaacccaatt 1381 gtgccagcct ggtttggtaa ttgacctcga gacgacctct ctgcccgttg tggtgatctc 1441 caacgtcagc cagctcccga gcggttgggc ctccatcctt tggtacaaca tgctggtggc 1501 ggaacccagg aatctgtcct tcttcctgac tccaccatgt gcacgatggg ctcagctttc 1561 agaagtgctg agttggcagt tttcttctgt caccaaaaga ggtctcaatg tggaccagct 1621 gaacatgttg ggagagaagc ttcttggtcc taacgccagc cccgatggtc tcattccgtg 1681 gacgaggttt tgtaaggaaa atataaatga taaaaatttt cccttctggc tttggattga 1741 aagcatccta gaactcatta aaaaacacct gctccctctc tggaatgatg ggtgcatcat 1801 gggcttcatc agcaaggagc gagagcgtgc cctgttgaag gaccagcagc cggggacctt 1861 cctgctgcgg ttcagtgaga gctcccggga aggggccatc acattcacat gggtggagcg 1921 gtcccagaac ggaggcgaac ctgacttcca tgcggttgaa ccctacacga agaaagaact 1981 ttctgctgtt actttccctg acatcattcg caattacaaa gtcatggctg ctgagaatat 2041 tcctgagaat cccctgaagt atctgtatcc aaatattgac aaagaccatg cctttggaaa 2101 gtattactcc aggccaaagg aagcaccaga gccaatggaa cttgatggcc ctaaaggaac 2161 tggatatatc aagactgagt tgatttctgt gtctgaagtt cacccttcta gacttcagac 2221 cacagacaac ctgctcccca tgtctcctga ggagtttgac gaggtgtctc ggatagtggg 2281 ctctgtagaa ttcgacagta tgatgaacac agtatagagc atgaattttt ttcatcttct 2341 ctggcgacag ttt

The polypeptide sequence of human Stat 2 is depicted in SEQ ID NO: 7. The nucleotide sequence of human Stat 2 is shown in SEQ ID NO: 8. Sequence information related to Stat 2 is accessible in public databases by GenBank Accession numbers AAA98760 (for protein) and U18671.1 (for nucleic acid).

SEQ ID NO: 7 is the human wild type amino acid sequence corresponding to Stat2 (residues 1-851): 1 MAQWEMLQNL DSPFQDQLHQ LYSHSLLPVD IRQYLAVWIE DQNWQEAALG SDDSKATMLF 61 FHFLDQLNYE CGRCSQDPES LLLQHNLRKF CRDIQPFSQD PTQLAEMIFN LLLEEKRILI 121 QAQRAQLEQG EPVLETPVES QQHEIESRIL DLRAMMEKLV KSISQLKDQQ DVFCFRYKIQ 181 AKGKTPSLDP HQTKEQKILQ ETLNELDKRR KEVLDASKAL LGRLTTLIEL LLPKLEEWKA 241 QQQKACIRAP IDHGLEQLET WFTAGAKLLF HLRQLLKELK GLSCLVSYQD DPLTKGVDLR 301 NAQVTELLQR LLHRAFVVET QPCMPQTPHR PLILKTGSKF TVRTRLLVRL QEGNESLTVE 361 VSIDRNPPQL QGFRKFNILT SNQKTLTPEK GQSQGLIWDF GYLTLVEQRS GGSGKGSNKG 421 PLGVTEELHI ISFTVKYTYQ GLKQELKTDT LPVVIISNMN QLSIAWASVL WFNLLSPNLQ 481 NQQFFSNPPK APWSLLGPAL SWQFSSYVGR GLNSDQLSML RNKLFGQNCR TEDPLLSWAD 541 FTKRESPPGK LPFWTWLDKI LELVHDHLKD LWNDGRIMGF VSRSQERRLL KKTMSGTFLL 601 RFSESSEGGI TCSWVEHQDD DKVLIYSVQP YTKEVLQSLP LTEIIRHYQL LTEENIPENP 661 LRFLYPRIPR DEAFGCYYQE KVNLQERRKY LKHRLIVVSN RQVDELQQPL ELKPEPELES 721 LELELGLVPE PELSLDLEPL LKAGLDLGPE LESVLESTLE PVIEPTLCMV SQTVPEPDQG 841 PVSQPVPEPD LPCDLRHLNT EPMEIFRNCV KIEEIMPNGD PLLAGQNTVD EVYVSRPSHF 781 YTDGPLMPSD F SEQ ID NO: 8 is the human wild type nucleotide sequence corresponding to Stat2 (nucleotides 1-18648), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame: 1 tcaagatcag cctgggcaac atggcgaaac cccgtctcta caataaatac aaaaaaatta 61 tcctggcgga gttatgcacg ttgtagtccc aactacctgg gaggctgagg cgggagaatc 121 acctgagcct gggaggtcga ggctgcagcg agccgagatc ggccgctgca ttccagcctg 181 ggtgacagag cgagaccatg tctcaaaaaa taaaaattaa aaaaaaattg ttttcattac 241 ctcagccctc ctcttcctat cccaaggcgt cgaaattccg gtcccacccc ttcccatgga 301 gcccttggcg tctccaggct cctcaagcta gtttcggttc cgggctcacg cgcgggttct 361 cgaaaatcag ctgtttcagt cttgggctag tccactaatt ggactcctcc cctcgtagaa 421 agtgcctact tgaacttctc caccaatcgc tgaagctgca ggtgtggttt cggctcagct 481 tgtcccgccc tggcggaggg gcggagttgc ggcggcgcca gtgagctcgc agtctgggaa 541 gggcttgact gaatggcagc cagtgtcggg gtggcggctg ggaatggggg ccgctccgga 601 cttccgctgc caactacaag ggggcgggtc cgaggggggt tagccgaagt tgtaggcggg 661 gcgcgaggtt ctagtacccg agctcatact agggacggga agtcgcgacc agagccattg 721 gagggcgcgg ggactgcaac cctaatcagg tacgggccct gagagggtgt gctggggtag 781 gggtgggggt gagagtgaga gttcctccga gggaagggcg actggcccag gggttacccc 841 ctggagaggg tagcttcctt ccccagattg aaataggagc tgtcgcctgc tcggtcctcg 901 atcttcttct gtccagccta tctccctaac cctaatgccc ctctcccaaa actgccctgc 961 agcttccgag acccggaatc tggcattgtt atgttggttc ggtatctgac gtttttccct 1021 ctgctctgca ttatttttta tcttcaccaa aaaacgatgt tcaaagatag ataaatctaa 1081 aaacaaagat agataaatct attacccttg tttcgtaaaa agtataagct actgaaagat 1141 gaaacgattg cctaaggtca cacacaaaat tcagttcatt tcagaaaagc ttcttgagtg 1201 caaaatatgt gcctaagaat gagagataat gagaaaaaat tgtttcagcc ccttaacctc 1261 agtgtttgca atccatttgg ggagaccagg ttttttgttt ttgttttcat atttgaatct 1321 ttgctgactt gctcctttaa tatcagacac ttaaatcctc agatgggact catcatattt 1381 tttttgagat ggaatcttca ctatgttgct caagcttggt ctgcaactcc tggctcaagc 1441 catcctctcg tcttgttggg cctctcgtct tgtgggcctg cacaaagtgc tgggattaca 1501 ggcatgagcc attcatgccc tgggcgcacc ttggattgcg atgtgtgtgt gttgtgaagc 1561 tttttttttt ggtatcataa aagcaataca gatacatagt tttaaaaatc aagcagctac 1621 taaaagagtt aaaatgaaaa tagcccctcc caatccctcc cttgttcctg ctggaggtag 1681 aaaggcagct gatgttattc atgttagtag aagactctcc caccccaagc atttctcttt 1741 attttgtaat aaaatcatgt gaccttttta gaccacaaat atgcatgaat tctgttctgt 1801 taggctcagg ctgcaacaag ataagtttca gtttcctaaa tagacaccag ctggcagtga 1861 gcagggaaca gtggggagaa agatgcatgg gacagcctgc ttggtgacag gcaaaaaccg 1921 gtttgttgtt cttttagaga cagagtcttg ctttgtcacc caggctggag tgtagtgatg 1981 tgatctctgc ttactgcaac cctgcctctg ggtacaagcc attctcctgc ctcagcctct 2041 tgagtagctg ggattacagg caacaatttt aagtgaagtg aagtttcagg atctcgagca 2101 aagttgtata acctataatc atattcaaga ttcacaggtc ataaacgtgt catattcttg 2161 ggattgagcg acccattgca cagcatttag atgtgcttct agaatggagc tcctccttcc 2221 tatatggagg gcagtttata tggtgtactt acctgaccac caaaaagatt tggctctaaa 2281 aaagcttcag gtggccgggc atggtggttc acccctgtaa tccagcactt tgggaggcag 2341 gtgggcagat cacctgaggt cagaagttca gacagctgga catatggtga aacctcatct 2401 ctactaaaaa tacaaaaatt agactgggca tggtagtggg cgcctgtaat cccagctagt 2461 cgggaggctg aggcaggaga atcccttcaa ctcggacggc agagtttgca gtgaggccga 2521 gatcgtgtca ctgcagtcca gcctgggtga cagagcaaga ctccatctca aaaaaagtaa 2581 aaaaaaaaaa aagaaaaaaa aaagcttcag agccagcagg gatcatgctg taataaatac 2641 ttaacatcaa cactgatctt taaatgcttt agcacaatca aatataaata acaaacacac 2701 acataaatgc aaaataaatg aattagggag atagatgaaa taagattgtg gaaatagtaa 2761 tgtttgttaa agctggatgg tgatccttgt actattcact ctactctagt gtgtatttga 2821 aaattaccat taggctggtt atggtggctc atgcctgtta atcccggcat tttggaaggc 2881 tgaggcaggc ggattacttg agctcaggag tttagagtct gcctgggcaa catggcaaaa 2941 tcccatctct acaaaaaatt agctggcatg atggcacact cctgtagtcc cagctccttg 3001 aggggctgag gcagagaatg gcttgaacct gagaggctaa agctgcagtg agccaagatc 3061 atgccactgc actccagcct gggtgaccaa gtgagaccct gtctcaaaaa aaaaaaaaaa 3121 aaaaagaaaa gaaaattccc attaaagcac aaaggcccac ttattgaagc tattaaaata 3181 caggttgggg ccggctgggc atcgcgtcac gcctgtaatc ccagcacttt ggaaggccga 3241 ggtaggcgag tcacgagttc aggagatcga gaccatcctg gctaacacgg tgaaacccca 3301 tctctactaa aaatacaaaa aaaaaaatca gccgggcatg gtggcgggag cctatagtcc 3361 cagctactcg ggaggctgag gcaggagaat ggcatgagcc cgggaggcgg agcttgcagt 3421 gagccaaaat cacaccactg cactccagcc tgggcaacag atcgagactc catctgaaga 3481 aaaaaaaaat acaggttggg accacagtgg ctcatgcctg taatcctagt actttgggag 3541 tccgaagtag gtggatcacc tgaggtcagg actttgagac cagcctggcc aacatggcaa 3601 aaccccatct ctactaaaaa atatacaaaa attagctggg cgtggtggtg ggtgcctgta 3661 atcccagcta ctcaggaggc tgaggcagaa gaatcacaac aaccaggggg atggtggttg 3721 caatgagcca agatcatctc cacttcactc cggcccaggc aaaagagtga gagtcatctt 3781 aaaaaaaaaa aaaaaaaaaa aaaaaaaata cagattaggc attcctaatc tgaaaaattt 3841 ggctccaaaa tgctccagtc gagcatttcc tttgagtgtc atgtgggtgc tcaaaaagtt 3901 agatttttgg accattttca gatttcagag ttttggatta gggatgctcg actggtaagt 3961 aatcgagata ttccaaaaat ctggacaaat ctgaaatcca aaatgcttgg aatagcagat 4021 actcaactgg tagcactccc tggaagaata tgcaccaaac tgatagcagt ggttaccttc 4081 tggtgaggag gggaaagaac caagattagc agtaggatca acatatattt taatgttttc 4141 tgtattttta ttacttgtat aatttaaaca ttttaaatta gtaataatga acaatcatga 4201 aactatggat gatttagtcc agcaaaatat ccaattggga accctcatcc ttctgcagag 4261 cccaa atg gc gcagtgggaa atgctgcaga atcttgacag cccctttcag gatcagctgc 4321 accagcttta ctcgcacagc ctcctgcctg tggacattcg acagtacttg gctgtctgga 4381 ttgaagacca gaactggtga ggccttcagg aagttggggg aatgaaaaag gtggccttcc 4441 acttctgggc ccccgggatc ctggaatcat taatggcagg aaggggttgg aaagcctcag 4501 gactacagta acactgcaga gacactaata cttcttattc ctggtcccag gcaggaagct 4561 gcacttggga gtgatgattc caaggctacc atgctattct tccacttctt ggatcagctg 4621 aactatgagt gtggccgttg cagccaggac ccagagtcct tgttgctgca gcacaatttg 4681 cggaaattct gccgggacat tcaggtactt ggaacggttg ggagtgatgg ggtagcactg 4741 ggagcagagc atagaggagt aaggtttgga gaatagaata gtacctggag gtggcaaggg 4801 agacgggaac aaatgtgggg aaaggaggac agagtctgga cttggggaat cactagcaga 4861 gagaagggtt gcatatacgt gacactgttg ggaggatgct atggtgaaaa gacaaagggc 4921 taagaacccc gaaggaggag gaaatactgt ggacattggt ggggagggtc tagggcaata 4981 ggtcattgag agtggttgaa ttggatcaat cctttctgtt tacctttctg ttagcccttt 5041 tcccaggatc ctacccagtt ggctgagatg atctttaacc tccttctgga agaaaaaaga 5101 attttgatcc aggctcagag ggcccaattg gtgaggacaa ttcagtggta atgttggaaa 5161 ctcctgaagt agagaggaac catggaaagg actcagggag ttgtctcaga acaggatccc 5221 cccgacatcc tgtggtataa tttcaggcct gaacttaagg catgaaaggc cagagttaaa 5281 acgtgctcag agcctctttt ttcaggaaca aggagagcca gttctcgaaa cacctgtgga 5341 gagccagcaa catgagattg aatcccggat cctggattta agggctatga tggaggttag 5401 tagatgtggt aggagttagg gttgacagtg ttcagcctaa cacctccctg agaagcagcc 5461 tcatcggggt cctctcccct ctgcagaagc tggtaaaatc catcagccaa ctgaaagacc 5521 agcaggatgt cttctgcttc cgatataaga tccaggccaa aggtaggaag cacattgagg 5581 ggctggagaa agataagtgc ctgctgagaa gccggagctg gaagtgaaca ggagaaagct 5641 ccgatgagca gtagtcactg tcagacacac cccactgact acagtcctgc tgccgtgcaa 5701 agctggaatc gtgctttgtg gaggctgagc tggaggtgac agctgagaga cagtaaattg 5761 ttgaggaaat gcatggaaaa ctaacagtgt tttatttgag ggggtgtctg gtccaagatg 5821 accacttcag aatttgcctg gagggtccca caggtgcctg tgctttgctt ggtttccctt 5881 tcttcctccg ccacaaaatt cctccttcct gactctgact gagaccccag tcaggaagga 5941 gaggaaagaa cccctggact gactcctgtt cccaccatcc agggaagaca ccctctctgg 6001 acccccatca gaccaaagag cagaagattc tgcaggaaac tctcaatgaa ctggacaaaa 6061 ggagaaaggt gggaggcagc agaacagaac atgtgggcaa caaggacctg aaaaaatgag 6121 ggatgttggg aaccctggta atctagcgct ggcttctttc tttcttcatc cccagttggg 6181 tggtggaggg tgaaagggag agatgctcaa cactcacatt atctctttcc caggaggtgc 6241 tggatgcctc caaagcactg ctaggccgat taactaccct aatcgagcta ctgctgccaa 6301 agttggagga gtggaaggcc cagcagcaaa aagcctgcat cagagctccc attgaccacg 6361 ggttggaaca gctggagaca tggtgagagg taccacccca accctcgtcc tcgccatgcg 6421 ctgtgatttg taagttgcag tgccctgcat atagcaagag atactgttct ctatttgtct 6481 ctgctcccca gaatagagcc ctgctccctg cctgactgca gctctattct gcctcctcag 6541 cctcaccacg cagggaagcc cagaagtccc agtctccttc agggaaagga atgaattaac 6601 ccacaatctg gttttgcttc ttttttttaa tcacccagaa atatatatat atgtattttt 6661 tttttactgc aacgaataca atgacaagaa aggaagggaa ggaaggaagg aagagaaaat 6721 tacctattac ctagcttatt aaacaaaaat ggaatcatat tgtccatact attttgaaat 6781 ccatggggtt ttttttaagc ttaacagtat tttatatata tatatatata tatatatata 6841 tatatatata tatatatata tatatttttt tttttttttt tttttttttt ttttgagacg 6901 gagtctctct ctgttccctg gctggcggag cggagtcggc acgatctcag ctcactgcaa 6961 cttccaactc ccacggttca agccaattct cctgtctcag cctcccgagc ctgggattac 7021 caggcacaca ccagcctggc tagttttttt gattttttag tagagacgat gtttctccat 7081 gttggccagg ctggtctcaa actcctgact tcaggtgatc cacccaactt gggctcccaa 7141 agtgctggga ttacaggcgt gacgaccatg cccggccaac agtatattat atttatccat 7201 gttatttctt atgtccacac aacagtcccc tatatggtgg taacataatt taattaatga 7261 actcctattt tcagctattt aggttatttt caatttcttg ttaccttttg ccaggaaacg 7321 tatattttat ggtaattata ttgtgttgta gaaaaatcac tagtctagtc caacttgctt 7381 gaaaaatagc tactttttaa ctattttctc atttaaaaat ttattataat ttagtctttt 7441 agaaatatac caggccaggc atggcgtctc atgcctgtta tcctagtact ttggaaggct 7501 gaggacggag gatcacttca gtcttggggt ttgagaccag cccgggaaac ataacaagac 7561 cccatctcta caaaaaaaaa aaattgtttt taattaggca tgtccgacac agtggctcac 7621 acatgtggcc agcactgtgg gaaggccaag gtgggtggat cacttgaggg tcaggagttc 7681 aagaccagcc tggccaatgt ggtgaaaccc catctctact aaaaatacaa aaatttgcca 7741 ggtgtggtgg cgcatgcctg tattcccagc tactcaggag gctaaggcag gaaatcactt 7801 gaactcggag gcagaggttg cagtgagctg tgacaatgcc actgtactcc agcctgggtg 7861 acagagcgag ctccgtctca aaaaaaaaaa aaaaagatta ggcatggtgg cacacgcctg 7921 tagaccctag ctactcagga ggctgaggtg ggaggattgc ttgagcccag gtgttggagg 7981 ctgcagtgag ccatgattat accactgtag tccagcctgg acaacagaac gagaccctgt 8041 ctctaaaagt atatatgtac acataccata atacccagct actgaggagg ctgaggcaga 8101 aagagtgctt gagtccagga gtttgatgtc agcctgagca atatagcaag accctcacct 8161 cttaaaaaaa tttaaagtag attaaaaaaa taccacaatt gctcaggtag attaaaaaaa 8221 taccacaatt gctcaggtag attattgaaa aacaggcata tagtacttat ggtacaggac 8281 cagcatgcat gcatgcatgc attgattgat tgattgattg attgattgag acagggtctc 8341 tctctgtctc ccaggctgga gtgcctggcc ttaagtgatc tgcccacctt tgcttcccaa 8401 agtgctgaga ttacaggtgt gagccaccat gtcagctggc gaggcttttt aaaagatagt 8461 tccaagtgtt acagctcttt taggatttgt ctagcaggct ttcaggtttt tgccagaaac 8521 cacccccacc cccaccaaaa aaaaaaaaaa aaaaaagata tgtacaagtt cccagatagt 8581 gttcccaact gaatctattt ctcatgtgta gtgtatggtt gttttcctgt caccacattg 8641 ctgattatta ttatttttaa ttatagagac agtaaagtac agtagttaaa aatgtgagtt 8701 ggggctgggt gcagtggctc acacctgtaa tcccagcact ttgggaggcc aaggtgggcg 8761 gatcacctga ggtcaggagt tcaagaccag cttggccaac atggcaaaac cccgtctcga 8821 ctaaaaatat atatatataa gttagccggg cgtggtggca acattacctg taatcccagc 8881 tactcgggag gccaacaggc aggagaatct cttgaatcca ggaggtggag gttgcagtga 8941 gccagatcac accattgcac tccagcctgg atgacaagag agtgagactg tctaaaaaaa 9001 aaaaacaaag tgtgagttgt acaatgagac tgcctgggat cacatacaag cttcatccct 9061 tactagttgt attgacccta aagcaagtca ctaacctttc tgtgccctcc agttttatca 9121 tctgtaatgt ggggaaaata atagtacctg cctcagaggg ttgttttgag gattaaatgc 9181 attaatatgt ggaaagggct taatataagt tgtacatagc atatgaaaac tgttatgtta 9241 aatctattag cagttttata tgtgaaaata gctttgattt tcatttcttg gattatgaat 9301 catgttgaat aatcctttat atgcttcctg gattcttttt ttttcttccc cccagtcagt 9361 ttctgactct tctcatattt atagagagat cttggaacct ggatggggga atccaggaaa 9421 ctcatggatt ccttcttcct gaattttatc acccaggttc acagctggag caaagctgtt 9481 gtttcacctg aggcagctgc tgaaggagct gaagggactg agttgcctgg ttagctatca 9541 ggatgaccct ctgaccaaag gggtggacct acgcaacgcc caggtcacag agttgctaca 9601 gcgtctgctc cacaggtcta gaggccaggc aggaaccctg ggggaaagaa ggaacaaggg 9661 aagccattct tacacatact gagctatata ttctctccac acctctctct cctcgagcct 9721 ttgtggtaga aacccagccc tgcatgcccc aaactcccca tcgacccctc atcctcaaga 9781 ctggcagcaa gttcaccgtc cgaacaaggt tggcattcca gaactcattc ccacttcctt 9841 tttccaaccc tgccactgtg tattttctgg ctttacagct actgcccact cttggctttt 9901 tcagtctttc ctgaatctcc ctacctcgtt gataccccat cgtcctcttt ttcaaacacc 9961 tagcctatac aaaagccgac tccgaccaca tttccctata ccccttgact tccccaggct 10021 gctggtgaga ctccaggaag gcaatgagtc actgactgtg gaagtctcca ttgacaggta 10081 aattggagca ggtgaagggt ggccaggaca cgggctgctg gggtggagga gatactcact 10141 cttcacaaca gggccctagg gctatatcct tcctccttcc aatcctacct cacagaaatt 10201 ataattcatt tcttttgttg aacacttact ttgtgacatg cagcatgtca gctactcatt 10261 taattgtcac accaacccca tgaataaact attaccagtg cactgtacaa acaaagatac 10321 aggcttagag agactgatta catctcttct caaggccaca tagctagtga gctcaagtcg 10381 ggtttgaacc gaggtctgtc tgatcccaaa gacgaaactc ctaacttcca tactcttttg 10441 cccaatgatt ttttttaaat ttatttcttt tcaggaatcc tcctcaatta caagggtagg 10501 tgcttgacaa ggacactgca aacatctgta cagtgtatga cctgcagaac cgggggattt 10561 gggaaatgga caaagggaga tggcgagatc tgaaatggaa gtggaacttc agtttttttt 10621 ttttctgctg agtttttaca ataattccat tccttgtctc catgtatctt cctcctggaa 10681 cagcttccgg aagttcaaca ttctgacttc aaaccagaaa actttgaccc ccgagaaggg 10741 gcagagtcag ggtttgattt gggactttgg ttacctggta agaatagttt gtgacctatg 10801 cttttattac tatttttatt ttttcgagac ggagtctcac tctgtccccc aggctggagt 10861 gcagtggtgc catcttggct cacaggaacc tccgccctcc ccggttcaag caattcttct 10921 gtctcagcct cctgagtacg tagagctata ggcagcacac caccatgccc ggctaatttt 10981 tgtattttta gtagagatag ggtttcacca tattggtcgg gctggtctcg aactcctgac 11041 ctcaggtgat ccgacccgcc tcagcctccc aaagtgctgg gatcacaggc atgagccacc 11101 atagctggcc tgcttttagt ccaaaggaac aggggttggg ggaagttccc agggcttgag 11161 aggtcttgaa gccaaacagg ggttccaggg agactagggt gcccactctg gcattttctc 11221 tccttccctt caattcacag actctggtgg agcaacgttc aggtggttca ggaaagggca 11281 gcaataaggt gagatctgga cagaggactc gaggcagggg gagcttgcca aagagccttc 11341 tgatgactat gtctttgcct gtcccagagg ggccactagg tgtgacagag gaactgcaca 11401 tcatcagctt cacggtcaaa tatacctacc agggtctgaa gcaggagctg aaagtgagtg 11461 aaaatggagg gcaaggagag agaaagcagc tttggaagaa ggcataagaa ggggataaac 11521 agaagcctct tggggagggt tagcactcct ttcctctaac aaatacctgc agctagaaac 11581 atcacatccc tctctgtgac tcctgtcttc tccccacaca cggacaccct ccctgtggtg 11641 attatttcca acatgaacca gctctcaatt gcctgggctt cagttctctg gttcaatttg 11701 ctcagcccaa accttcaggt aggggagtgg ggccgacagg tcccggcgcg agagcagggg 11761 tgtggaagct tggtgtgata ggttgcttct gagccagcct acactgctcc cacccctgca 11821 gaaccagcag ttcttctcca acccccccaa ggccccctgg agcttgctgg gccctgctct 11881 cagttggcag ttctcctcct atgttggccg aggcctcaac tcagaccagc tgagcatgct 11941 gagaaacaag ctgttcggta cagatttcct tttctctcag cctttcccca gccttagtct 12001 tttctgtccc tctgtcctat ctatcccagg acccctggct tccctcacat atctgtggct 12061 atctgtccca cagggcagaa ctgtaggact gaggatccat tattgtcctg ggctgacttc 12121 actaaggtaa ctccctgaat cctgtggagc tgctggatct agccccacat tccaaatact 12181 ggccttccca cgtgccctcc ttccctacac cagaggcaac tcctcagctt ttgctacctt 12241 tccattcctc cagcgagaga gccctcctgg caagttacca ttctggacat ggctggacaa 12301 aattctggag ttggtacatg accacctgaa ggatctctgg aatgatgggt aaggccttgg 12361 tcacccttcc ctcatgggct tgtgcttccg ggcttgagag tggagtctct gcaccctcac 12421 gtggcaagca gggagagaga gcaaagcacg gtgcaggcca cgtctcctca catttgttaa 12481 gaataataag gccgggtgtg gtggctcaca cctgtaatcc cagcactttg ggaggccgag 12541 gcgggcggat catgaggtca ggagatcgag accatcctgg ctaacacggt gaaaccccgt 12601 ctctactcta aaaatacaaa aaattagccg ggcgtggagg cagacaccct gtagtcccag 12661 ctactcagga ggctgaggca ggaaaatggc gtgaacctgg gagatggagc ttgcagtgag 12721 ccgagattgc gtcactgccc tccagccttg gggtgacgta gcaagactcc gtctcaaaaa 12781 aaaaaaaaaa aaacaaccaa taatagccat aaacagtgtt tttgtgaagc actcctacat 12841 tccagagctt gatgggtgct cttcattaat tctctcatct catccttaca accatgctga 12901 gtggtgggtt ttgccagctt catttcatgt gaggaaactg agtttcagag aagttaaaga 12961 acttacccaa gggacacagt tgatattcaa atccaggcct atgtgactcc aagcccatgc 13021 tctttccacc acactgccta ccaacttgtg tagcatttgg cttttaaaag tgctattcat 13081 gaccaggcac gatggctcac gccttgtaat cccagcattt tgggaggccg aggtgggtgg 13141 atcacctgag gtcaggagtt tgagaccagc ctggccaaca tggcgaaacc ccatctctat 13201 taaaaataca aaaattagcc gggtgtggtg gtgggcgcct gtaatcccag ctactcagga 13261 ggctgaggag gagaatcgct tgaatttagg agagaaggtt acagtgagcc aagatcgtgc 13321 cattgcactc cagcctgggt gacagagcaa gactctgtct caaaacaaaa ccaaaaaaaa 13381 gtgctatttg tggccaggcg tggttgctca tgcctgtaat cctagcattt ttggggaggc 13441 tgaggagtac agatcacttg agcccaggag ttcaaaacta ccctgggcca cgtggtgaaa 13501 ccccaaaccc cgtctctacg aaaaatacaa aagttagcca ggatgggtgg tgtgcacctg 13561 tggtcccagc tactctggag gctgagaggt ggggaagatt gcttgagccc gggaggtcga 13621 ggtggcagtg agctgtgatc atgccactat tctccagcct gggtgacaga atacaccctg 13681 tctccctgtc tcccagaaaa aaaaaaaagt gctgttcatc tgtgtgatct cactgaatct 13741 tcgtacttca aaccctcgga aggtggctat tgtcagcaaa gtgaagtgac ttgtaaaaga 13801 taaaaaaaag ctaagtggca gggcttggtc caaagcctgg attccaaacc tgggctgttt 13861 ctccatacaa ggggagcagg gaggcagggg cctggggggg cagggtgttg ggcggtgtca 13921 cacgtgacac actgtgctcc agacgcatca tgggctttgt gagtcggagc caggagcgcc 13981 ggctgctgaa gaagaccatg tctggcacct ttctactgcg cttcagtgaa tcgtcagaag 14041 ggggcattac ctgctcctgg gtggagcacc aggatgatgg tagctgctct gccctgccat 14101 tcccacagcc tctcctttct gcctggctct cctctggccc ctctgcctgc cttgcttcgc 14161 tggctctgaa ctgaatgctc agtggtttgg gactgggcag ccagagagtc agagagctcc 14221 aaggcccggc ctcttccctc aagcccgcct gttcctgcat tcactctcca gacaaggtgc 14281 tcatctactc tgtgcaaccg tacacgaagg aggtgctgca gtcactcccg ctgactgaaa 14341 tcatccgcca ttaccagttg ctcactgagg agaatatacc tgaaaaccca ctgcgcttcc 14401 tctatccccg aatcccccgg gatgaagctt ttgggtgcta ctaccaggag aaaggtggga 14461 atcgttgaca tacttcattg ctagattgca gagatctacc agacatccat agatcccact 14521 ccttccttta aagcatggga aaactgatat ctagaggaat taagggattc gtccatggga 14581 tactgctggt tactatgggg atgagactgc caggaccatc tgcactaggg gaaaacctca 14641 ggctatatgt ctggcccact gatcttctct gcttcttgta tatgttcctc acagttaatc 14701 tccaggaacg gaggaaatac ctgaaacaca ggctcattgt ggtctctaat agagtgagat 14761 atgaactgtt cattcatcct ccctaatcct tattggctct gcttcagtga atcgtcaaaa 14821 gggggcatta ccttctcctg ggtggagcac caggatgatg gtcagctgct ctgccctgcc 14881 attcccacag cctctccttt ctgccttctc ctaagctgcc cctattccag tctccccagc 14941 cttccctccc tcctagcccc actctagttt tttctggttc tagtctctcc tatctcatat 15001 ttttctgctg ccatccttag gttgtctcca caggggtttc tggataataa tgatcataat 15061 cactggtgtt aaggggtacc tacttgatgc aagcatggag cttttttttt ttccagacag 15121 ggttttgttc tgtcgcccag gctggagtgc agtggtgtga tcctggctca ctgcagcctc 15181 gacctcctga gctcaagcaa tacaggcatg catcaccaaa ctcagctaat tttttttgta 15241 ttttttgtag agatggggtc ttaccatgtt gacgcatcag gctgttctga actcctggac 15301 tcaagcaatc cacccacctt ggcctcccaa aagtcaggga ttacaggcgt gcgaccacac 15361 cccgcatata tatatttttt tttttttttt tttttttttt tttttgagac agggtctctg 15421 ttatccaggc tggagttgca gtggataata tgactacgag ccttgaccta ggggttgaag 15481 caatgctcct gcctcagcca ccaagtgctg agactacagg cacacgccaa tctacactca 15541 atcacactca gctaattttt taaatttttt gtagggatgg ggtatcactg tgtttgccca 15601 ggctggtctt gaactcctgg cctcaagcag tctcctgcct tggcctccca aattgccggg 15661 attgtaggaa tgagccatgg cacttggctg ggggatagaa tttttttttt tttttttttt 15721 tttttttttt ttgagacagt ctcactctca ttgcccgggc tggagtgcag tggtgcaatt 15781 tcagctcact gcaacctctg cctcccaggc tcaagcaatt ctcctgcctc agcctataga 15841 gtagctggga ttacaggcga gcgccaccca tgcctggtta atttttgttt tttttttgag 15901 acagagtctc gccctgttgc ccaggctgga gtgcagtggc acgatctcag ctcactgcaa 15961 cctctgcctc ccaggctcaa gcaattctcc tgcctcagcc tcctgagtac tgggactaca 16021 agcgcgcaca accaccacac ctggtaattt ttgtattttt agtagagaca gggttttacc 16081 atattggcca ggctggtctc aaactcctga cctcatgatc cgacccacct tggcctccca 16141 aagtgcaggg attacaggcg tgagcctctg cacccggcct aacttttgta tttttagtag 16201 aaacagggtt tcaccatgtt ggccaggctg gtcatgagct cctggcctca agtgatctgc 16261 ccgcctcagc ctcccaaagt gcttggatta caggtgtgag ccacctggcc tgagagttta 16321 ttatgcgcca ggcactaggc aaatggtttg catttatttt ctcattttat tgaatctaca 16381 aaatagtcct gtgaagtaaa cactgttact gttttcagct aaggaactgg atttagagta 16441 gtcaagtttt gtacctaagg tacgtggcta atgatacagg tctgttagat tccgtagccc 16501 tgattttaac caccctactg cctctcaaga attactaggt attgttctca tttatagatg 16561 ataaatctga ggctcagaaa agttaggcca cttgcctaag gtcccccagc caggattcaa 16621 actccaggag gcctgattcc aaacccatgc tctttagccc tccgccctac tgccttctta 16681 gactagcttc tgcttattct accattcctg atttcatttg aaccactgag ccctgcccct 16741 ttgtctgtct ttgggtatcc aggcaggtgg atgaactgca acaaccgctg gagcttaagc 16801 cagagccaga gctggagtca ttagagctgg aactagggct ggtgccagag ccagagctca 16861 gcctggactt agagccactg ctgaaggcag ggctggatct ggggccagag ctagagtctg 16921 tgctggagtc cactctggag cctgtgatag agcccacact atgcatggta tcacaaacag 16981 tgccagagcc agaccaagga cctgtatcac agccagtgcc agagccagat ttgccctgtg 17041 atctgagaca tttgaacact gagccaatgg aaagtaagtg atgagatgga gtggcacaca 17101 ttccctttcc tacctcttct ccctctccca ttacagaaaa agctgaactc caagctcctc 17161 attggagaga ggtccatctg tgattccttt ttttaggaat tacacatgcc ttcccccacc 17221 tccctgctct ttcatcccac aagttcccac tcaggctctt cccaggcctt tcctgccatc 17281 ctccctccct tgggctgctg ggttgggaac tcctaactaa gatcggggcc tcacttttct 17341 ctctggatta cctagtcttc agaaactgtg taaagattga agaaatcatg ccgaatggtg 17401 acccactgtt ggctggccag aacaccgtgg atgaggttta cgtctcccgc cccagccact 17461 tctacactga tggacccttg atgccttctg acttctagga accacatttc ctctgttctt 17521 ttcatatctc tttgcccttc ctactcctca tagcatgata ttgttctcca aggatgggaa 17581 tcaggcatgt gtcccttcca agctgtgtta actgttcaaa ctcaggcctg tgtgactcca 17641 ttggggtgag aggtgaaagc ataacatggg tacagagggg acaacaatga atcagaacag 17701 atgctgagcc ataggtctaa ataggatcct ggaggctgcc tgctgtgctg ggaggtatag 17761 gggtcctggg ggcaggccag ggcagttgac aggtacttgg agggctcagg gcagtggctt 17821 ctttccagta tggaaggatt tcaacatttt aatagttggt taggctaaac tggtgcatac 17881 tggcattggc cttggtgggg agcacagaca caggatagga ctccatttct ttcttccatt 17941 ccttcatgtc taggataact tgctttcttc tttcctttac tcctggctca agccctgaat 18001 ttcttctttt cctgcagggg ttgagagctt tctgccttag cctaccatgt gaaactctac 18061 cctgaagaaa gggatggata ggaagtagac ctctttttct taccagtctc ctcccctact 18121 ctgcccccta agctggctgt acctgttcct cccccataaa atgatcctgc caatctaatg 18181 tgagtgtgaa gtttgcacac tagtttatgc tacctagtct ccactttctc aatgcttagg 18241 agacagatca ctcctggagg ctggggatgg taggattgct ggggattttt ttttttttaa 18301 agagggtctc actctgttgc ccaggctaga gtgcaatggt gcaatcacag ctcactgcag 18361 cctcaacctc ctgggttcaa gcaatcctcc tacctcagcc tcctgggtag ctagcaccat 18421 ggcatcgcca ccatgcccta tttttttttt ttaaagacag ggtcttgcta tattgcccag 18481 gctggtcttg aactgggctc aagtgatcct cacgccttgc ctcccaaagt gctgggatta 18541 taggcatgag ccactgtgct tggccaggat tttttttttt ttttttttga gatggagttt 18601 ctctcttgtt gtccaggctg gagtgcaatg gtgtgatccg gggaattc

Protein variants can include amino acid sequence modifications. For example, amino acid sequence modifications fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions can include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. These variants ordinarily are prepared by site-specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.

Nucleic acid sequences comprising a gene, such as a Jak1, Jak2, Jak3, Stat 1, and/or Stat 2 gene, that encodes a polypeptide can be synthesized, in whole or in part, using chemical methods known in the art. Alternatively, a polypeptide, such as a Jak1, Jak2, Jak3, Stat 1, and/or Stat 2, can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques. Protein synthesis can either be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of Jak1, Jak2, Jak3, Stat 1, and/or Stat 2 polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.

As used herein, a Jak1, Jak2, Jak3, Stat 1, or Stat 2 “molecule” can be a nucleic acid which encodes a polypeptide that exhibits Jak1, Jak2, Jak3, Stat 1, and/or Stat 2 activity, or a polypeptide or peptidomimetic that exhibits Jak1, Jak2, Jak3, Stat 1, and/or Stat 2 activity. For example, a Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule can include the human Jak1, Jak2, Jak3, Stat 1, or Stat 2 protein, or a variant thereof, such as a fragment thereof, that exhibits Jak1, Jak2, Jak3, Stat 1, and/or Stat 2 activity. Jak1, Jak2, Jak3, Stat 1, and/or Stat 2 activity can encompass signaling events by way of type I cytokine receptors (e.g., IL-2, IL-4, IL-7, IL-9, IL-15, IL-21) that use the common gamma chain (γc). For example, Jak1, Jak2, Jak3, Stat 1, and/or Stat 2 activity can be a signal transduced in response to its activation via tyrosine phosphorylation by interleukin receptors.

The nucleic acid can be any type of nucleic acid, including genomic DNA, complementary DNA (cDNA), synthetic or semi-synthetic DNA, as well as any form of corresponding RNA. For example, a Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule can comprise a recombinant nucleic acid encoding human Jak1, Jak2, Jak3, Stat 1, or Stat 2 protein. In one embodiment, a Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule can comprise a non-naturally occurring nucleic acid created artificially (such as by assembling, cutting, ligating or amplifying sequences). A Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule can be double-stranded. A Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule can be single-stranded. The Jak1, Jak2, Jak3, Stat 1, and/or Stat 2 molecules of the invention can be obtained from various sources and can be produced according to various techniques known in the art. For example, a nucleic acid that is a Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule can be obtained by screening DNA libraries, or by amplification from a natural source. The Jak1, Jak2, Jak3, Stat 1, and/or Stat 2 molecules can be produced via recombinant DNA technology and such recombinant nucleic acids can be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof. Non-limiting examples of a Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule that is a nucleic acid. Another example of a Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule is a fragment of a nucleic acid, wherein the fragment exhibits Jak1, Jak2, Jak3, Stat 1, and/or Stat 2 activity. A Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule of this invention also encompasses variants of the human nucleic acid encoding the Jak1, Jak2, Jak3, Stat 1, or Stat 2 protein, or variants of the human Jak1, Jak2, Jak3, Stat 1, or Stat 2 proteins that exhibit Jak1, Jak2, Jak3, Stat 1, and/or Stat 2 activity. A Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule can also include a fragment of the human Jak1, Jak2, Jak3, Stat 1, or Stat 2 nucleic acid which encodes a polypeptide that exhibits Jak1, Jak2, Jak3, Stat 1, and/or Stat 2 activity. A Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule can encompass a fragment of the human Jak1, Jak2, Jak3, Stat 1, or Stat 2 protein that exhibits Jak1, Jak2, Jak3, Stat 1, and/or Stat 2 activity.

A Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule can also encompass Jak1, Jak2, Jak3, Stat 1, or Stat 2 ortholog genes, which are genes conserved among different biological species such as humans, dogs, cats, mice, and rats, that encode proteins (for example, homologs (including splice variants), mutants, and derivatives) having biologically equivalent functions as the human-derived protein (such as a Jak3 protein). Jak1, Jak2, Jak3, Stat 1, or Stat 2 orthologs include any mammalian ortholog of Jak1, Jak2, Jak3, Stat 1, or Stat 2 inclusive of the ortholog in humans and other primates, experimental mammals (such as mice, rats, hamsters and guinea pigs), mammals of commercial significance (such as horses, cows, camels, pigs and sheep), and also companion mammals (such as domestic animals, e.g., rabbits, ferrets, dogs, and cats).

The Jak1, Jak2, Jak3, Stat 1, and Stat 2 variants can comprise, for instance, naturally-occurring variants due to allelic variations between individuals (e.g., polymorphisms), mutated alleles related to alopecia areata, or alternative splicing forms. In one embodiment, a Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule is a nucleic acid variant of the nucleic acid having the sequence shown in herein, wherein the variant has a nucleotide sequence identity to a corresponding sequence disclosed herein of about 65%, about 75%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. In one embodiment, a Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule encompasses any portion of about 8 consecutive nucleotides of a corresponding sequence disclosed herein. In one embodiment, the fragment can comprise about 15 nucleotides, about 20 nucleotides, or about 30 nucleotides of a corresponding sequence disclosed herein. Fragments include all possible nucleotide lengths between about 8 and 100 nucleotides, for example, lengths between about 15 and 100, or between about 20 and 100.

The invention further provides for nucleic acids that are complementary to a nucleic acid encoding a Jak1, Jak2, Jak3, Stat 1, or Stat 2 protein. Such complementary nucleic acids can comprise nucleic acid sequences, which hybridize to a nucleic acid sequence encoding a Jak1, Jak2, Jak3, Stat 1, or Stat 2 protein under stringent hybridization conditions. Non-limiting examples of stringent hybridization conditions include temperatures above 30° C., above 35° C., in excess of 42° C., and/or salinity of less than about 500 mM, or less than 200 mM. Hybridization conditions can be adjusted by the skilled artisan via modifying the temperature, salinity and/or the concentration of other reagents such as SDS or SSC.

In one embodiment, a Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule comprises a protein or polypeptide encoded by a Jak1, Jak2, Jak3, Stat 1, or Stat 2 nucleic acid sequence, such as the corresponding sequence disclosed herein. In another embodiment, the polypeptide can be modified, such as by glycosylations and/or acetylations and/or chemical reaction or coupling, and can contain one or several non-natural or synthetic amino acids. An example of a Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule is the polypeptide having the amino acid sequence disclosed herein. In another embodiment, a Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule can be a fragment of a Jak1, Jak2, Jak3, Stat 1, or Stat 2 protein. For example, the Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule can encompass any portion of about 8 consecutive amino acids of the corresponding sequence disclosed herein. The fragment can comprise about 10 amino acids, a least about 20 amino acids, about 30 amino acids, about 40 amino acids, a least about 50 amino acids, about 60 amino acids, or about 75 amino acids of the corresponding sequence disclosed herein. Fragments include all possible amino acid lengths between about 8 and 100 about amino acids, for example, lengths between about 10 and 100 amino acids, between about 15 and 100 amino acids, between about 20 and 100 amino acids, between about 35 and 100 amino acids, between about 40 and 100 amino acids, between about 50 and 100 amino acids, between about 70 and 100 amino acids, between about 75 and 100 amino acids, or between about 80 and 100 amino acids.

In certain embodiments, the Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule includes variants of the human Jak1, Jak2, Jak3, Stat 1, or Stat 2 protein (comprising the corresponding sequence disclosed herein). Such variants can include those having at least from about 46% to about 50% identity to the corresponding sequence disclosed herein, or having at least from about 50.1% to about 55% identity to the corresponding sequence disclosed herein, or having at least from about 55.1% to about 60% identity to SEQ ID NO: 1, or having from about 60.1% to about 65% identity to the corresponding sequence disclosed herein, or having from about 65.1% to about 70% identity to the corresponding sequence disclosed herein, or having at least from about 70.1% to about 75% identity to the corresponding sequence disclosed herein, or having at least from about 75.1% to about 80% identity to the corresponding sequence disclosed herein, or having at least from about 80.1% to about 85% identity to the corresponding sequence disclosed herein, or having at least from about 85.1% to about 90% identity to the corresponding sequence disclosed herein, or having at least from about 90.1% to about 95% identity to the corresponding sequence disclosed herein, or having at least from about 95.1% to about 97% identity to the corresponding sequence disclosed herein, or having at least from about 97.1% to about 99% identity to the corresponding sequence disclosed herein.

Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions can be single residues, but can occur at a number of different locations at once. In one non-limiting embodiment, insertions can be on the order of about from 1 to about 10 amino acid residues, while deletions can range from about 1 to about 30 residues. Deletions or insertions can be made in adjacent pairs (for example, a deletion of about 2 residues or insertion of about 2 residues). Substitutions, deletions, insertions, or any combination thereof can be combined to arrive at a final construct. The mutations cannot place the sequence out of reading frame and should not create complementary regions that can produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place.

Substantial changes in function or immunological identity are made by selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions that can produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation and/or glycosylation.

There can be minor variations in the amino acid sequences of the corresponding sequence disclosed herein for each of Jak1, Jak2, Jak3, Stat 1, and/or Stat 2. The variations in the amino acid sequence can be when the sequence maintains about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 90%, about 95%, or about 99% identity to the corresponding sequence disclosed herein. For example, conservative amino acid replacements can be utilized. Conservative replacements are those that take place within a family of amino acids that are related in their side chains, wherein the interchangeability of residues have similar side chains.

Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) a group of amino acids having aliphatic-hydroxyl side chains, such as serine and threonine; (ii) a group of amino acids having amide-containing side chains, such as asparagine and glutamine; (iii) a group of amino acids having aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; (iv) a group of amino acids having aromatic side chains, such as phenylalanine, tyrosine, and tryptophan; and (v) a group of amino acids having sulfur-containing side chains, such as cysteine and methionine. Useful conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, glutamic-aspartic, and asparagine-glutamine.

For example, the replacement of one amino acid residue with another that is biologically and/or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Substitutional or deletional mutagenesis can be employed to insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr). Deletions of cysteine or other labile residues also can be desirable. Deletions or substitutions of potential proteolysis sites, e.g. Arg, is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.

In another embodiment, the Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule encompasses a peptidomimetic which exhibits Jak1, Jak2, Jak3, Stat 1, or Stat 2 activity. A peptidomimetic is a small protein-like chain designed to mimic a peptide that can arise from modification of an existing peptide in order to protect that molecule from enzyme degradation and increase its stability, and/or alter the molecule's properties (e.g., modifications that change the molecule's stability or biological activity). These modifications involve changes to the peptide that cannot occur naturally (such as altered backbones and the incorporation of non-natural amino acids). Drug-like compounds can be developed from existing peptides. A peptidomimetic can be a peptide, partial peptide, or non-peptide molecule that mimics the tertiary binding structure or activity of a selected native peptide or protein functional domain (e.g., binding motif or active site). These peptide mimetics include recombinantly or chemically modified peptides.

In one embodiment, a Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule comprising the corresponding sequence disclosed herein, variants of such, or fragments thereof, can be modified to produce peptide mimetics by replacement of one or more naturally occurring side chains of the 20 genetically encoded amino acids (or D amino acids) with other side chains. This can occur, for instance, with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4, 5-, 6-, to 7-membered heterocyclics. For example, proline analogs can be made in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members. Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic. Heterocyclic groups can contain one or more nitrogen, oxygen, and/or sulphur heteroatoms. Examples of such groups include the furazanyl, ifuryl, imidazolidinyl imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g. 1-piperazinyl), piperidyl (e.g. 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g. 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g. thiomorpholino), and triazolyl. These heterocyclic groups can be substituted or unsubstituted. Where a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl. Peptidomimetics can also have amino acid residues that have been chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other moieties. For example, peptidomimetics can be designed and directed to amino acid sequences encoded by a Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule comprising the corresponding sequence disclosed herein.

A variety of techniques are available for constructing peptide mimetics with the same or similar desired biological activity as the corresponding native but with more favorable activity than the peptide with respect to solubility, stability, and/or susceptibility to hydrolysis or proteolysis (see, e.g., Morgan & Gainor, Ann. Rep. Med. Chem. 24,243-252, 1989). Certain peptidomimetic compounds are based upon the amino acid sequence of the peptides of the invention. Peptidomimetic compounds can be synthetic compounds having a three-dimensional structure (i.e. a peptide motif) based upon the three-dimensional structure of a selected peptide. The peptide motif provides the peptidomimetic compound with the desired biological activity, wherein the binding activity of the mimetic compound is not substantially reduced, and is often the same as or greater than the activity of the native peptide on which the mimetic is modeled. Peptidomimetic compounds can have additional characteristics that enhance their therapeutic application, such as increased cell permeability, greater affinity and/or avidity and prolonged biological half-life. Peptidomimetic design strategies are readily available in the art (see, e.g., Ripka & Rich (1998) Curr. Op. Chem. Biol. 2:441-452; Hruby et al. (1997) Curr. Op. Chem. Biol. 1:114-119; Hruby & Balse, (2000) Curr. Med. Chem. 9:945-970).

Cell Transfection

A eukaryotic expression vector can be used to transfect cells in order to produce proteins encoded by nucleotide sequences of the vector. Mammalian cells (such as isolated cells from the hair bulb; for example dermal sheath cells and dermal papilla cells) can contain an expression vector (for example, one that contains a gene encoding a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein or polypeptide) via introducing the expression vector into an appropriate host cell via methods known in the art.

A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed polypeptide encoded by a gene, such as a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, Va. 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.

An exogenous nucleic acid can be introduced into a cell via a variety of techniques known in the art, such as lipofection, microinjection, calcium phosphate or calcium chloride precipitation, DEAE-dextran-mediated transfection, or electroporation. Electroporation is carried out at approximate voltage and capacitance to result in entry of the DNA construct(s) into cells of interest (such as cells of the end bulb of a hair follicle, for example dermal papilla cells or dermal sheath cells). Other transfection methods also include modified calcium phosphate precipitation, polybrene precipitation, liposome fusion, and receptor-mediated gene delivery.

Cells that will be genetically engineered can be primary and secondary cells obtained from various tissues, and include cell types which can be maintained and propagated in culture. Non-limiting examples of primary and secondary cells include epithelial cells (for example, dermal papilla cells, hair follicle cells, inner root sheath cells, outer root sheath cells, sebaceous gland cells, epidermal matrix cells), neural cells, endothelial cells, glial cells, fibroblasts, muscle cells (such as myoblasts) keratinocytes, formed elements of the blood (e.g., lymphocytes, bone marrow cells), and precursors of these somatic cell types.

Vertebrate tissue can be obtained by methods known to one skilled in the art, such a punch biopsy or other surgical methods of obtaining a tissue source of the primary cell type of interest. In one embodiment, a punch biopsy or removal can be used to obtain a source of keratinocytes, fibroblasts, endothelial cells, or mesenchymal cells (for example, hair follicle cells or dermal papilla cells). In another embodiment, removal of a hair follicle can be used to obtain a source of fibroblasts, keratinocytes, endothelial cells, or mesenchymal cells (for example, hair follicle cells or dermal papilla cells). A mixture of primary cells can be obtained from the tissue, using methods readily practiced in the art, such as explanting or enzymatic digestion (for examples using enzymes such as pronase, trypsin, collagenase, elastase dispase, and chymotrypsin). Biopsy methods have also been described in United States Patent Application Publication 2004/0057937 and PCT application publication WO 2001/32840, and are hereby incorporated by reference.

Primary cells can be acquired from the individual to whom the genetically engineered primary or secondary cells are administered. However, primary cells can also be obtained from a donor, other than the recipient, of the same species. The cells can also be obtained from another species (for example, rabbit, cat, mouse, rat, sheep, goat, dog, horse, cow, bird, or pig). Primary cells can also include cells from an isolated vertebrate tissue source grown attached to a tissue culture substrate (for example, flask or dish) or grown in a suspension; cells present in an explant derived from tissue; both of the aforementioned cell types plated for the first time; and cell culture suspensions derived from these plated cells. Secondary cells can be plated primary cells that are removed from the culture substrate and replated, or passaged, in addition to cells from the subsequent passages. Secondary cells can be passaged one or more times. These primary or secondary cells can contain expression vectors having a gene that encodes a protein of interest (for example, a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein or polypeptide).

Cell Culturing

Various culturing parameters can be used with respect to the host cell being cultured. Appropriate culture conditions for mammalian cells are well known in the art (Cleveland W L, et al., J Immunol Methods, 1983, 56(2): 221-234) or can be determined by the skilled artisan (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B. D., eds. (Oxford University Press: New York, 1992)). Cell culturing conditions can vary according to the type of host cell selected. Commercially available medium can be utilized. Non-limiting examples of medium include, for example, Minimal Essential Medium (MEM, Sigma, St. Louis, Mo.); Dulbecco's Modified Eagles Medium (DMEM, Sigma); Ham's F10 Medium (Sigma); HyClone cell culture medium (HyClone, Logan, Utah); RPMI-1640 Medium (Sigma); and chemically-defined (CD) media, which are formulated for various cell types, e.g., CD-CHO Medium (Invitrogen, Carlsbad, Calif.).

The cell culture media can be supplemented as necessary with supplementary components or ingredients, including optional components, in appropriate concentrations or amounts, as necessary or desired. Cell culture medium solutions provide at least one component from one or more of the following categories: (1) an energy source, usually in the form of a carbohydrate such as glucose; (2) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine; (3) vitamins and/or other organic compounds required at low concentrations; (4) free fatty acids or lipids, for example linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that can be required at very low concentrations, usually in the micromolar range.

The medium also can be supplemented electively with one or more components from any of the following categories: (1) salts, for example, magnesium, calcium, and phosphate; (2) hormones and other growth factors such as, serum, insulin, transferrin, and epidermal growth factor; (3) protein and tissue hydrolysates, for example peptone or peptone mixtures which can be obtained from purified gelatin, plant material, or animal byproducts; (4) nucleosides and bases such as, adenosine, thymidine, and hypoxanthine; (5) buffers, such as HEPES; (6) antibiotics, such as gentamycin or ampicillin; (7) cell protective agents, for example pluronic polyol; and (8) galactose. In one embodiment, soluble factors can be added to the culturing medium.

The mammalian cell culture that can be used with the present invention is prepared in a medium suitable for the type of cell being cultured. In one embodiment, the cell culture medium can be any one of those previously discussed (for example, MEM) that is supplemented with serum from a mammalian source (for example, fetal bovine serum (FBS)). In another embodiment, the medium can be a conditioned medium to sustain the growth of epithelial cells or cells obtained from the hair bulb of a hair follicle (such as dermal papilla cells or dermal sheath cells). For example, epithelial cells can be cultured according to Barnes and Mather in Animal Cell Culture Methods (Academic Press, 1998), which is hereby incorporated by reference in its entirety. In a further embodiment, epithelial cells or hair follicle cells can be transfected with DNA vectors containing genes that encode a polypeptide or protein of interest (for example, a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein or polypeptide). In other embodiments of the invention, cells are grown in a suspension culture (for example, a three-dimensional culture such as a hanging drop culture) in the presence of an effective amount of enzyme, wherein the enzyme substrate is an extracellular matrix molecule in the suspension culture. For example, the enzyme can be a hyaluronidase. Epithelial cells or hair follicle cells can be cultivated according to methods practiced in the art, for example, as those described in PCT application publication WO 2004/044188 and in U.S. Patent Application Publication No. 2005/0272150, or as described by Harris in Handbook in Practical Animal Cell Biology: Epithelial Cell Culture (Cambridge Univ. Press, Great Britain; 1996; see Chapter 8), which are hereby incorporated by reference.

A suspension culture is a type of culture wherein cells, or aggregates of cells (such as aggregates of DP cells), multiply while suspended in liquid medium. A suspension culture comprising mammalian cells can be used for the maintenance of cell types that do not adhere or to enable cells to manifest specific cellular characteristics that are not seen in the adherent form. Some types of suspension cultures can include three-dimensional cultures or a hanging drop culture. A hanging-drop culture is a culture in which the material to be cultivated is inoculated into a drop of fluid attached to a flat surface (such as a coverglass, glass slide, Petri dish, flask, and the like), and can be inverted over a hollow surface. Cells in a hanging drop can aggregate toward the hanging center of a drop as a result of gravity. However, according to the methods of the invention, cells cultured in the presence of a protein that degrades the extracellular matrix (such as collagenase, chondroitinase, hyaluronidase, and the like) will become more compact and aggregated within the hanging drop culture, for degradation of the ECM will allow cells to become closer in proximity to one another since less of the ECM will be present. See also International PCT Publication No. WO2007/100870, which is incorporated by reference.

Cells obtained from the hair bulb of a hair follicle (such as dermal papilla cells or dermal sheath cells) can be cultured as a single, homogenous population (for example, comprising DP cells) in a hanging drop culture so as to generate an aggregate of DP cells. Cells can also be cultured as a heterogeneous population (for example, comprising DP and DS cells) in a hanging drop culture so as to generate a chimeric aggregate of DP and DS cells. Epithelial cells can be cultured as a monolayer to confluency as practiced in the art. Such culturing methods can be carried out essentially according to methods described in Chapter 8 of the Handbook in Practical Animal Cell Biology: Epithelial Cell Culture (Cambridge Univ. Press, Great Britain; 1996); Underhill C B, J Invest Dermatol, 1993, 101(6):820-6); in Armstrong and Armstrong, (1990) J Cell Biol 110:1439-55; or in Animal Cell Culture Methods (Academic Press, 1998), which are all hereby incorporated by reference in their entireties.

Three-dimensional cultures can be formed from agar (such as Gey's Agar), hydrogels (such as matrigel, agarose, and the like; Lee et al., (2004) Biomaterials 25: 2461-2466) or polymers that are cross-linked. These polymers can comprise natural polymers and their derivatives, synthetic polymers and their derivatives, or a combination thereof. Natural polymers can be anionic polymers, cationic polymers, amphipathic polymers, or neutral polymers. Non-limiting examples of anionic polymers can include hyaluronic acid, alginic acid (alginate), carageenan, chondroitin sulfate, dextran sulfate, and pectin. Some examples of cationic polymers, include but are not limited to, chitosan or polylysine. (Peppas et al., (2006) Adv Mater. 18: 1345-60; Hoffman, A. S., (2002) Adv Drug Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Ann NY Acad Sci 944: 62-73). Examples of amphipathic polymers can include, but are not limited to collagen, gelatin, fibrin, and carboxymethyl chitin. Non-limiting examples of neutral polymers can include dextran, agarose, or pullulan. (Peppas et al., (2006) Adv Mater. 18: 1345-60; Hoffman, A. S., (2002) Adv Drug Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Ann NY Acad Sci 944: 62-73).

Cells suitable for culturing according to methods of the invention can harbor introduced expression vectors, such as plasmids. The expression vector constructs can be introduced via transformation, microinjection, transfection, lipofection, electroporation, or infection. The expression vectors can contain coding sequences, or portions thereof, encoding the proteins for expression and production. Expression vectors containing sequences encoding the produced proteins and polypeptides, as well as the appropriate transcriptional and translational control elements, can be generated using methods well known to and practiced by those skilled in the art. These methods include synthetic techniques, in vitro recombinant DNA techniques, and in vivo genetic recombination which are described in J. Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. and in F. M. Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.

Obtaining and Purifying Polypeptides

A polypeptide molecule encoded by a gene, such as a Jak 1, Jak 2, Jak3, Stat 1, or Stat 2 gene, or a variant thereof, can be obtained by purification from human cells expressing a protein or polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1, or Stat 2 gene via in vitro or in vivo expression of a nucleic acid sequence encoding a Jak 1, Jak 2, Jak3, Stat 1, or Stat 2 protein or polypeptide; or by direct chemical synthesis.

Detecting Polypeptide Expression. Host cells which contain a nucleic acid encoding a Jak 1, Jak 2, Jak3, Stat 1, or Stat 2 protein or polypeptide, and which subsequently express a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1, or Stat 2 gene, can be identified by various procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a nucleic acid encoding a Jak 1, Jak 2, Jak3, Stat 1, or Stat 2 protein or polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments of nucleic acids encoding a Jak 1, Jak 2, Jak3, Stat 1, or Stat 2 protein or polypeptide. In one embodiment, a fragment of a nucleic acid of a Jak 1, Jak 2, Jak3, Stat 1, or Stat 2 gene can encompass any portion of at least about 8 consecutive nucleotides of the corresponding sequence disclosed herein. In another embodiment, the fragment can comprise at least about 10 consecutive nucleotides, at least about 15 consecutive nucleotides, at least about 20 consecutive nucleotides, or at least about 30 consecutive nucleotides of the corresponding sequence disclosed herein. Fragments can include all possible nucleotide lengths between about 8 and about 100 nucleotides, for example, lengths between about 15 and about 100 nucleotides, or between about 20 and about 100 nucleotides. Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1, or Stat 2 gene to detect transformants which contain a nucleic acid encoding a Jak 1, Jak 2, Jak3, Stat 1, or Stat 2 protein or polypeptide.

Protocols for detecting and measuring the expression of a polypeptide encoded by a gene, such as a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, using either polyclonal or monoclonal antibodies specific for the polypeptide are well established. Non-limiting examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a polypeptide encoded by a gene, such as a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, can be used, or a competitive binding assay can be employed.

Labeling and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Methods for producing labeled hybridization or PCR probes for detecting sequences related to nucleic acid sequences encoding a protein, such as Jak 1, Jak 2, Jak3, Stat 1 or Stat 2, include, but are not limited to, oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, nucleic acid sequences encoding a polypeptide encoded by a gene, such as a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, and/or magnetic particles.

Expression and Purification of Polypeptides. Host cells transformed with a nucleic acid sequence encoding a polypeptide, such as Jak 1, Jak 2, Jak3, Stat 1 or Stat 2, can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. Expression vectors containing a nucleic acid sequence encoding a polypeptide, such as Jak 1, Jak 2, Jak3, Stat 1 or Stat 2, can be designed to contain signal sequences which direct secretion of soluble polypeptide molecules encoded by a gene, such as a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, or a variant thereof, through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound a polypeptide molecule encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or a variant thereof.

Other constructions can also be used to join a gene sequence encoding a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). Including cleavable linker sequences (i.e., those specific for Factor Xa or enterokinase (Invitrogen, San Diego, Calif.)) between the purification domain and a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene also can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene and 6 histidine (SEQ ID NO:111) residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by immobilized metal ion affinity chromatography, while the enterokinase cleavage site provides a means for purifying the polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene.

A Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 polypeptide can be purified from any human or non-human cell which expresses the polypeptide, including those which have been transfected with expression constructs that express a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein. A purified Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein can be separated from other compounds which normally associate with a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene in the cell, such as certain proteins, carbohydrates, or lipids, using methods practiced in the art. Non-limiting methods include size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.

Chemical Synthesis. Nucleic acid sequences comprising a gene, such as a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, that encodes a polypeptide can be synthesized, in whole or in part, using chemical methods known in the art. Alternatively, a polypeptide, such as Jak 1, Jak 2, Jak3, Stat 1 or Stat 2, can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques. Protein synthesis can either be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule. In one embodiment, a fragment of a nucleic acid sequence that comprises a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene can encompass any portion of at least about 8 consecutive nucleotides of the corresponding sequence disclosed herein. In one embodiment, the fragment can comprise at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, or at least about 30 nucleotides of the corresponding sequence disclosed herein. Fragments include all possible nucleotide lengths between about 8 and about 100 nucleotides, for example, lengths between about 15 and about 100 nucleotides, or between about 20 and about 100 nucleotides.

A Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 fragment can be a fragment of a protein, such as Jak 1, Jak 2, Jak3, Stat 1 or Stat 2. For example, the Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 fragment can encompass any portion of at least about 8 consecutive amino acids of the corresponding sequence disclosed herein. The fragment can comprise at least about 10 consecutive amino acids, at least about 20 consecutive amino acids, at least about 30 consecutive amino acids, at least about 40 consecutive amino acids, a least about 50 consecutive amino acids, at least about 60 consecutive amino acids, at least about 70 consecutive amino acids, or at least about 75 consecutive amino acids of the corresponding sequence disclosed herein. Fragments include all possible amino acid lengths between about 8 and 100 about amino acids, for example, lengths between about 10 and about 100 amino acids, between about 15 and about 100 amino acids, between about 20 and about 100 amino acids, between about 35 and about 100 amino acids, between about 40 and about 100 amino acids, between about 50 and about 100 amino acids, between about 70 and about 100 amino acids, between about 75 and about 100 amino acids, or between about 80 and about 100 amino acids.

A synthetic peptide can be substantially purified via high performance liquid chromatography (HPLC). The composition of a synthetic polypeptide of Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 can be confirmed by amino acid analysis or sequencing. Additionally, any portion of an amino acid sequence comprising a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.

Identifying Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 Modulating Compounds

The invention provides methods for identifying compounds which can be used for controlling and/or regulating hair growth (for example, hair density) or hair pigmentation in a subject. Since the invention has provided the identification of the genes listed herein as genes associated with a hair loss disorder, the invention also provides methods for identifying compounds that modulate the expression or activity of a gene and/or protein of Jak 1, Jak 2, Jak3, Stat 1 or Stat 2. In addition, the invention provides methods for identifying compounds which can be used for the treatment of a hair loss disorder. The invention also provides methods for identifying compounds which can be used for the treatment of hypotrichosis (for example, hereditary hypotrichosis simplex (HHS)). Non-limiting examples of hair loss disorders include: androgenetic alopecia, Alopecia areata, telogen effluvium, alopecia areata, alopecia totalis, and alopecia universalis. The methods can comprise the identification of test compounds or agents (e.g., peptides (such as antibodies or fragments thereof), small molecules, nucleic acids (such as siRNA or antisense RNA), or other agents) that can bind to a polypeptide molecule encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene and/or have a stimulatory or inhibitory effect on the biological activity of a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or its expression, and subsequently determining whether these compounds can regulate hair growth in a subject or can have an effect on symptoms associated with the hair loss disorders in an in vivo assay (i.e., examining an increase or reduction in hair growth).

As used herein, a “Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compound” refers to a compound that interacts with a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein or polypeptide and modulates its activity and/or its expression. The compound can either increase the activity or expression of a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. Conversely, the compound can decrease the activity or expression of a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. The compound can be a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 agonist or a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 antagonist (e.g., a Jak1 inhibitor, a Jak2 inhibitor, a Stat1 inhibitor, or a Stat2 inhibitor). Some non-limiting examples of Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds include peptides (such as peptide fragments comprising a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, or antibodies or fragments thereof), small molecules, and nucleic acids (such as siRNA or antisense RNA specific for a nucleic acid comprising a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene). Agonists of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein can be molecules which, when bound to a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, increase or prolong the activity of the Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein. Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 agonists include, but are not limited to, proteins, nucleic acids, small molecules, or any other molecule which activates a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein. Antagonists of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein can be molecules which, when bound to a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein decrease the amount or the duration of the activity of the Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein. Antagonists include proteins, nucleic acids, antibodies, small molecules, or any other molecule which decrease the activity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein.

The term “modulate,” as it appears herein, refers to a change in the activity or expression of a gene or protein of Jak 1, Jak 2, Jak3, Stat 1 or Stat 2. For example, modulation can cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein.

In one embodiment, a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compound can be a peptide fragment of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein that binds to the protein. For example, the Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 polypeptide can encompass any portion of at least about 8 consecutive amino acids of the corresponding sequence disclosed herein. The fragment can comprise at least about 10 consecutive amino acids, at least about 20 consecutive amino acids, at least about 30 consecutive amino acids, at least about 40 consecutive amino acids, at least about 50 consecutive amino acids, at least about 60 consecutive amino acids, or at least about 75 consecutive amino acids of the corresponding sequence disclosed herein. Fragments include all possible amino acid lengths between and including about 8 and about 100 amino acids, for example, lengths between about 10 and about 100 amino acids, between about 15 and about 100 amino acids, between about 20 and about 100 amino acids, between about 35 and about 100 amino acids, between about 40 and about 100 amino acids, between about 50 and about 100 amino acids, between about 70 and about 100 amino acids, between about 75 and about 100 amino acids, or between about 80 and about 100 amino acids. These peptide fragments can be obtained commercially or synthesized via liquid phase or solid phase synthesis methods (Atherton et al., (1989) Solid Phase Peptide Synthesis: a Practical Approach. TRL Press, Oxford, England). The Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 peptide fragments can be isolated from a natural source, genetically engineered, or chemically prepared. These methods are well known in the art.

A Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compound can be a protein, such as an antibody (monoclonal, polyclonal, humanized, chimeric, or fully human), or a binding fragment thereof, directed against a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. An antibody fragment can be a form of an antibody other than the full-length form and includes portions or components that exist within full-length antibodies, in addition to antibody fragments that have been engineered. Antibody fragments can include, but are not limited to, single chain Fv (scFv), diabodies, Fv, and (Fab′)₂, triabodies, Fc, Fab, CDR1, CDR2, CDR3, combinations of CDR's, variable regions, tetrabodies, bifunctional hybrid antibodies, framework regions, constant regions, and the like (see, Maynard et al., (2000) Ann. Rev. Biomed. Eng. 2:339-76; Hudson (1998) Curr. Opin. Biotechnol. 9:395-402). Antibodies can be obtained commercially, custom generated, or synthesized against an antigen of interest according to methods established in the art (Janeway et al., (2001) Immunobiology, 5th ed., Garland Publishing). In one embodiment, an antibody or binding fragment thereof is directed against SEQ ID NO: 1, 3, 5 or 7. Antibodies can be obtained commercially, custom generated, or synthesized against an antigen of interest according to methods established in the art (Janeway et al., (2001) Immunobiology, 5th ed., Garland Publishing). For example, antibodies directed to Jak1, Jak2, Stat1, or Stat2 can be obtained commercially from Abcam, Santa Cruz Biotechnology, Abnova Corp., BD Biosciences, Antigenix America Inc., etc. Human antibodies directed to either Jak1, Jak2, Stat1, or Stat2 (such as monoclonal, humanized, or chimeric antibodies) can be useful antibody therapeutics for use in humans.

Inhibition of RNA encoding a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene can effectively modulate the expression of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene from which the RNA is transcribed. Inhibitors are selected from the group comprising: siRNA; interfering RNA or RNAi; dsRNA; RNA Polymerase III transcribed DNAs; ribozymes; and antisense nucleic acids, which can be RNA, DNA, or an artificial nucleic acid.

Antisense oligonucleotides, including antisense DNA, RNA, and DNA/RNA molecules, act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the DNA sequence encoding a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene can be synthesized, e.g., by conventional phosphodiester techniques (Dallas et al., (2006) Med. Sci. Monit. 12(4):RA67-74; Kalota et al., (2006) Handb. Exp. Pharmacol. 173:173-96; Lutzelburger et al., (2006) Handb. Exp. Pharmacol. 173:243-59). Antisense nucleotide sequences include, but are not limited to: morpholinos, 2′-O-methyl polynucleotides, DNA, RNA and the like.

siRNA comprises a double stranded structure containing from about 15 to about 50 base pairs, for example from about 21 to about 25 base pairs, and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell. The siRNA comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions. The sense strand comprises a nucleic acid sequence which is substantially identical to a nucleic acid sequence contained within the target miRNA molecule. “Substantially identical” to a target sequence contained within the target mRNA refers to a nucleic acid sequence that differs from the target sequence by about 3% or less. The sense and antisense strands of the siRNA can comprise two complementary, single-stranded RNA molecules, or can comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded “hairpin” area. See also, McMnaus and Sharp (2002) Nat Rev Genetics, 3:737-47, and Sen and Blau (2006) FASEB J., 20:1293-99, the entire disclosures of which are herein incorporated by reference.

The siRNA can be altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, or modifications that make the siRNA resistant to nuclease digestion, or the substitution of one or more nucleotides in the siRNA with deoxyribo-nucleotides. One or both strands of the siRNA can also comprise a 3′ overhang. As used herein, a 3′ overhang refers to at least one unpaired nucleotide extending from the 3′-end of a duplexed RNA strand. For example, the siRNA can comprise at least one 3′ overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxyribonucleotides) in length, or from 1 to about 5 nucleotides in length, or from 1 to about 4 nucleotides in length, or from about 2 to about 4 nucleotides in length. For example, each strand of the siRNA can comprise 3′ overhangs of dithymidylic acid (“TT”) or diuridylic acid (“uu”).

siRNA can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector (for example, see U.S. Pat. Nos. 7,294,504 and 7,422,896, the entire disclosures of which are herein incorporated by reference). Exemplary methods for producing and testing dsRNA or siRNA molecules are described in U.S. Patent Application Publication No. 2002/0173478 to Gewirtz, U.S. Patent Application Publication No. 2007/0072204 to Hannon et al., and in U.S. Patent Application Publication No. 2004/0018176 to Reich et al., the entire disclosures of which are herein incorporated by reference.

In one embodiment, an siRNA directed to a human nucleic acid sequence comprising a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene can be generated against any one of SEQ ID NOS: 2, 4, 6 or 8. In another embodiment, an siRNA directed to a human nucleic acid sequence comprising a Jak1 gene can comprise any one of the sequences listed in Table 1. In another embodiment, an siRNA directed to a human nucleic acid sequence comprising a Stat1 gene can comprise any one of the sequences listed in Table 2. In another embodiment, an siRNA directed to a human nucleic acid sequence comprising a Jak3 gene can comprise any one of the sequences listed in Table 3.

In another embodiment, the siRNA directed to Jak1 is listed in Table 1.

TABLE 1 siRNA SEQUENCES for Jak1 (SEQ ID NOS: 9-32,  (SEQ ID NOS: 33-58, respectively, in respectively, in order of appearance) order of appearance) CCACATAGCTGATCTGAAA TCTGAAGAGAAGAAGATAA CGGGAAGCCTTAAGGAATA GCAAGAATGCATTGAACGA CTGAAGAGAAGAAGATAAA AAGAAAGATTGATGGACTT ACAAGAAGGATGAGGAGAA AAAATAAACTGAAGCGGAA CCAAGAAGACTGAGGTGAA ATGAGGAAATGCTGGGAAT CAGAATACGCCATCAATAA AAAGAAAGATTGATGGACT GCACAGAAGACGGAGGAAA GGACTTAGCCCTCAAATTT GCGATATATTCCAGAAACA ACACTGGACAGCTGAATAA TGAGCTACTTGGAGGATAA CTGAATAAATGCAGTATCT GAGTGAACACCAAGTGAAA AAGAATGCATTGAACGAAT CAAGAAGGATGAGGAGAAA CCAGTAACTTAGTGACACA CGGGAAGAGTGGAACAATT TTAACAAGCAGGACAACAA GCATTAACAAGCAGGACAA GGGAGCAGGTGGCTGTTAA TTTGGAAAGTAGAGAAGAA AAATCGAGATCTTAAGGAA GGAAAGTAGAGAAGAAAAT GGAAATGGTATTAAGCTCA GGAACCACGCTCTGGGAAA CCAGAATGTTTAATGCAAT TGATTGAGAAAGAGAGATT GGACAAGCTTTCAGAACCT ACATTACGGTGCTGAAATA ACACATAATGACAACCAAA GGGAAGAGTGGAACAATTT GCTTGGAGGTAGCTGGGTA AGAATATCATGGTGGAAGA CAGCAGAGGAACTGTGCAT GGAAATGTGTGTACTAAAA CCAGAAACATTGAATAAGT ATGAGAACATTGTGAAGTA CTGAAATATTTGAGACTTC CACAGAAGACGGAGGAAAT GTACAGAATACGCCATCAA GCTGGTGGCTACTAAGAAA GCACCGACTTTGACAACAT GAGAAGAAGATAAAAGTGA GGAAATTCAAAGTTGCCAA

In another embodiment, the siRNA directed to Stat1 is listed in Table 2.

TABLE 2 siRNA SEQUENCES for Stat1 (SEQ ID NOS: 59-81, (SEQ ID NOS: 82-108, respectively, in respectively, in order of appearance) order of appearance) AGAAAGAGCTTGACAGTAA TGAAATTGCAAGAGCTGAA AGGAATTGGAACAGAAATA AGAAATACACCTACGAACA CAGCATAACATAAGGAAAA CCCTAAAGGAACTGGATAT TCAGAAATGTGAAGGACAA GGAGGAATTGGAACASAAA GGCAAAGAGTGATCAGAAA CCTAAAGGAACTGGATATA GCACAGTGATGTTAGACAA AGCGTAATCTTCAGGATAA AGTCATGGCTGCTGAGAAT TCTGAAGGAAGAAAGGAAA GTATAGAGCATGAAATCAA GGAAGATTTACAAGATGAA GGTTATGTGTATAGAGCAT TGGCAAAGAGTGATCAGAA GAAAGGAAGCAGTTCACAA AGAGAAAGGAAGTAGTTCA GATGTGAATGAGAGAAATA TAATAGAGTTGCTGAATGT ACAGAGAACACGAGACCAA TGGAGGAATTGGAACAGAA AAGATGTGAATGAGAGAAA AGTTGAGACTGTTGGTGAA AGGACAAGGTTATGTGTAT GATAAAGATGTGAATGAGA CCTGATTAATGATGAACTA CATCGTTACTGAAGAGCTT ACACAAAAGTGATGAACAT TGAAGTATCTGATCCAAA GCAATTGAAAGAACAGAAA GCACAAGGTGGCAGGATGT CGAGAGCTGTCTAGGTTAA TGTCACAGCTGGATGATCA GAACATGACCCTATCACAA GAAAGAGCTTGACAGTAAA AGACAAACAGAAAGAGCTT TCAAGAGCCTGGAAGATTT GGACAAGGTTATGTGTATA TGGAAGATTTACAAGATGA TGACAATAAGAGAAAGGAA TGAGACTGTTGGTGAAATT GGAAGTAGTTCACAAAATA TTGCAAGAGCTGAATTATA GGATTTAGGAAGTTCAACA GGAACTTGATGGCCC AAA GGAACTGGATATATCAAGA

In another embodiment, the siRNA directed to Jak3 is listed in Table 3.

TABLE 3 siRNA SEQUENCES for Jak3 siRNA sequences TCAACTATCTGGAGGACAA (SEQ ID NO: 112) AGACAGAGGTGCTGCTGAA (SEQ ID NO: 113) GGTCCTTCACCAAGATTTA (SEQ ID NO: 114) CCTGGATCCTGCTAAGAAA (SEQ ID NO: 115) CGATCTTCGAGGAGAGACA (SEQ ID NO: 116) GGACAGACAACCAGATTTT (SEQ ID NO: 117) GGAAGCTGCAGGTGGTCAA (SEQ ID NO: 118) CCCAATACCAGCTGAGTCA (SEQ ID NO: 119)

RNA polymerase III transcribed DNAs contain promoters, such as the U6 promoter. These DNAs can be transcribed to produce small hairpin RNAs in the cell that can function as siRNA or linear RNAs that can function as antisense RNA. The Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compound can contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited. In addition, these forms of nucleic acid can be single, double, triple, or quadruple stranded. (see for example Bass (2001) Nature, 411, 428 429; Elbashir et al., (2001) Nature, 411, 494 498; and PCT Publication Nos. WO 00/44895, WO 01/36646, WO 99/32619, WO 00/01846, WO 01/29058, WO 99/07409, WO 00/44914).

A Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compound can be a small molecule that binds to a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein and disrupts its function, or conversely, enhances its function. Small molecules are a diverse group of synthetic and natural substances generally having low molecular weights. They can be isolated from natural sources (for example, plants, fungi, microbes and the like), are obtained commercially and/or available as libraries or collections, or synthesized. Candidate small molecules that modulate a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein can be identified via in silico screening or high-through-put (HTP) screening of combinatorial libraries (e.g., see Potyrailo et al., (2011) ACS Comb Sci. 13(6):579-633; Mensch et al., (2009) J Pharm Sci. 98(12):4429-68; Schnur (2008) Curr Opin Drug Discov Devel. 11(3):375-80; and Jhoti (2007) Ernst Schering Found Symp Proc. (3):169-85, each of which are hereby incorporated by reference in their entireties). Most conventional pharmaceuticals, such as aspirin, penicillin, and many chemotherapeutics, are small molecules, can be obtained commercially, can be chemically synthesized, or can be obtained from random or combinatorial libraries as described below (Werner et al., (2006) Brief Funct. Genomic Proteomic 5(1):32-6).

Non-limiting examples of Jak1/Jak2 inhibitors include: AG490 (Caceres-Cortes, Anticancer Agents Med Chem. 2008 October; 8(7):717-22); CYT387 (Pardanani et al., Leukemia. 2009 August; 23(8):1441-5; Monaghan et al., Leukemia. 2011 Jul. 26. doi: 10.1038/leu.2011.175. [Epub ahead of print]); SB1518 (William et al., J Med Chem. 2011 Jul. 14; 54(13):4638-58; Hart et al., Leukemia. 2011 Jun. 21. doi: 10.1038/leu.2011.148. [Epub ahead of print]); LY3009104 (INCB28050) (Incyte and Lilly); TG101348 (Wernig et al., Cancer Cell. 2008 April; 13(4):311-20; Pardanani et al., J Clin Oncol. 2011 Mar. 1; 29(7):789-96); and BMS-911543 (Purandare et al., Leukemia. 2011 Oct. 21. doi: 10.1038/leu.2011.292. [Epub ahead of print]), each of the references of which are incorporated by reference in their entireties.

JAK1/2 inhibitors in clinical development include a) INCB018424, topical and oral; 5 nM activity (Incyte); b) CEP-701 (Cephalon); and c) TG101348.

JAK3 inhibitors are currently in clinical trials in humans for the treatment of acute kidney transplant rejection and rheumatoid arthritis. Non-limiting examples of Jak3 inhibitors include: Janex 1 (WHI-P131) (Changelian et al., (2008) Blood, 111(4):2155-7); Uckun et al., (1999) Clin Cancer Res. 5(10):2954-62; Uckun et al., (2010) Arzneimittelforschung. 60(4):218-25), PF-956980 (Sigma Product #PZ0151 (St. Louis, Mo., sigmaaldrich.com/catalog/product/sigma/pz0151 ?lang=en&region=US)); Changelian et al., (2008) Blood, 111(4):2155-7), WHI-P154 (Calbiochem Product #420104-5MG (San Diego, Calif., emdbiosciences.com)); Changelian et al., (2008) Blood, 111(4):2155-7), VX-509 (oral from Vertex Pharmaceuticals, Cambridge Mass.; Fleischmann et al. (2011) Arthritis Rheum, 63:LB3; Fleischmann et al., (2012) Curr Opin Rheumatol. February 18, PMID: 22357358), JAK3 Inhibitor IV (ZM-39923) Calbiochem Product #420121-10MG (San Diego, Calif., emdbiosciences.com, WO1998022103), NSC114792 (Kim et al., (2010)Mol Cancer. 2010, 9:36), tofacitinib (CP690550) (Changelian et al., (2008) Blood, 111(4):2155-7; Vijayakrishnan et al. (2011) Trends Pharmacol Sci. 32(1):25-34; Fleischmann et al., (2012) Curr Opin Rheumatol. February 18, PMID: 22357358), and R348 (topical and oral from Rigel Pharmaceuticals, San Francisco Calif.; Deuse et al., (2008) Transplantation. 85(6):885-92; Vijayakrishnan et al. (2011) Trends Pharmacol Sci. 32(1):25-34), each of the references of which are incorporated by reference in their entireties.

Structures of JAK3 inhibitors useful for the invention include a) Janex 1, oral and topical; b) PF-956980, i.v. infusion; c) WHI-P154; d) ZM-39923; e) NSC114792; f) tofacitinib (CP690550), oral.

Non-limiting examples of JAK inhibtors (for example, Type I and Type II Jak Inhibitors) are discussed in O'Shea and Plenge (Immunity, 2012 Apr. 20; 36(4):542-50), LaFave and Levine (Trends Pharmacol Sci. 2012 November; 33(11):574-82), Kontzias et al, (Curr Opin Pharmacol. 2012 August; 12(4):464-70), Norman (Expert Opin Ther Pat. 2012 October; 22(10):1233-49), and Wilson (Expert Opin Ther Pat. 2010 May; 20(5):609-23), each of which are hereby incorporated by reference in their entireties.

Non-limiting examples of Stat inhibitors include: WP-1034 (Faderl et al., Anticancer Res. 2005 May-June; 25(3B):1841-50), fludarabine (Fludara, Berlex, Calif.), epigallocatechin-3-gallate (EGCG), and Hyperforin. Other compounds directed to Jak/Stat signaling are described in Ivanenkov et al., Mini Rev Med Chem. 2011 January; 11(1):55-78, the contents of which are incorporated by reference in its entirety.

Knowledge of the primary sequence of a molecule of interest, such as a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, and the similarity of that sequence with proteins of known function, can provide information as to the inhibitors or antagonists of the protein of interest in addition to agonists. Identification and screening of agonists and antagonists is further facilitated by determining structural features of the protein, e.g., using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for structure determination. These techniques provide for the rational design or identification of agonists and antagonists.

Test compounds, such as Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds, can be screened from large libraries of synthetic or natural compounds (see Wang et al., (2007) Curr Med Chem, 14(2):133-55; Mannhold (2006) Curr Top Med Chem, 6 (10):1031-47; and Hensen (2006) CurrMed Chem 13(4):361-76). Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), AMRI (Albany, N.Y.), ChemBridge (San Diego, Calif.), and MicroSource (Gaylordsville, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., (1996) Tib Tech 14:60).

Methods for preparing libraries of molecules are well known in the art and many libraries are commercially available. Libraries of interest in the invention include peptide libraries, randomized oligonucleotide libraries, synthetic organic combinatorial libraries, and the like. Degenerate peptide libraries can be readily prepared in solution, in immobilized form as bacterial flagella peptide display libraries or as phage display libraries. Peptide ligands can be selected from combinatorial libraries of peptides containing at least one amino acid. Libraries can be synthesized of peptoids and non-peptide synthetic moieties. Such libraries can further be synthesized which contain non-peptide synthetic moieties, which are less subject to enzymatic degradation compared to their naturally-occurring counterparts. For example, libraries can also include, but are not limited to, peptide-on-plasmid libraries, synthetic small molecule libraries, aptamer libraries, in vitro translation-based libraries, polysome libraries, synthetic peptide libraries, neurotransmitter libraries, and chemical libraries.

Examples of chemically synthesized libraries are described in Fodor et al., (1991) Science 251:767-773; Houghten et al., (1991) Nature 354:84-86; Lam et al., (1991) Nature 354:82-84; Medynski, (1994) BioTechnology 12:709-710; Gallop et al., (1994) J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., (1993) Proc. Natl. Acad Sci. USA 90:10922-10926; Erb et al., (1994) Proc. Natl. Acad Sci. USA 91:11422-11426; Houghten et al., (1992) Biotechniques 13:412; Jayawickreme et al., (1994) Proc. Natl. Acad Sci. USA 91:1614-1618; Salmon et al., (1993) Proc. Natl. Acad Sci. USA 90:11708-11712; PCT Publication No. WO 93/20242, dated Oct. 14, 1993; and Brenner et al., (1992) Proc. Natl. Acad Sci. USA 89:5381-5383.

Examples of phage display libraries are described in Scott et al., (1990) Science 249:386-390; Devlin et al., (1990) Science, 249:404-406; Christian, et al., (1992) J. Mol. Biol. 227:711-718; Lenstra, (1992) J. Immunol. Meth. 152:149-157; Kay et al., (1993) Gene 128:59-65; and PCT Publication No. WO 94/18318.

In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058; and Mattheakis et al., (1994) Proc. Natl. Acad Sci. USA 91:9022-9026.

As used herein, the term “ligand source” can be any compound library described herein, or tissue extract prepared from various organs in an organism's system, that can be used to screen for compounds that would act as an agonist or antagonist of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein. Screening compound libraries listed herein [also see U.S. Patent Application Publication No. 2005/0009163, which is hereby incorporated by reference in its entirety], in combination with in vivo animal studies, functional and signaling assays described below can be used to identify Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds that regulate hair growth or treat hair loss disorders.

Screening the libraries can be accomplished by any variety of commonly known methods. See, for example, the following references, which disclose screening of peptide libraries: Parmley and Smith, (1989) Adv. Exp. Med Biol. 251:215-218; Scott and Smith, (1990) Science 249:386-390; Fowlkes et al., (1992) BioTechniques 13:422-427; Oldenburg et al., (1992) Proc. Natl. Acad Sci. USA 89:5393-5397; Yu et al., (1994) Cell 76:933-945; Staudt et al., (1988) Science 241:577-580; Bock et al., (1992) Nature 355:564-566; Tuerk et al., (1992) Proc. Natl. Acad Sci. USA 89:6988-6992; Ellington et al., (1992) Nature 355:850-852; U.S. Pat. Nos. 5,096,815; 5,223,409; and 5,198,346, all to Ladner et al.; Rebar et al., (1993) Science 263:671-673; and PCT Pub. WO 94/18318.

Small molecule combinatorial libraries can also be generated and screened. A combinatorial library of small organic compounds is a collection of closely related analogs that differ from each other in one or more points of diversity and are synthesized by organic techniques using multi-step processes. Combinatorial libraries include a vast number of small organic compounds. One type of combinatorial library is prepared by means of parallel synthesis methods to produce a compound array. A compound array can be a collection of compounds identifiable by their spatial addresses in Cartesian coordinates and arranged such that each compound has a common molecular core and one or more variable structural diversity elements. The compounds in such a compound array are produced in parallel in separate reaction vessels, with each compound identified and tracked by its spatial address. Examples of parallel synthesis mixtures and parallel synthesis methods are provided in U.S. Ser. No. 08/177,497, filed Jan. 5, 1994 and its corresponding PCT published patent application WO95/18972, published Jul. 13, 1995 and U.S. Pat. No. 5,712,171 granted Jan. 27, 1998 and its corresponding PCT published patent application WO96/22529, which are hereby incorporated by reference.

In one non-limiting example, non-peptide libraries, such as a benzodiazepine library (see e.g., Bunin et al., (1994) Proc. Natl. Acad. Sci. USA 91:4708-4712), can be screened. Peptoid libraries, such as that described by Simon et al., (1992) Proc. Natl. Acad. Sci. USA 89:9367-9371, can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994), Proc. Natl. Acad. Sci. USA 91:11138-11142.

Computer modeling and searching technologies permit the identification of compounds, or the improvement of already identified compounds, that can modulate the expression or activity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein. Having identified such a compound or composition, the active sites or regions of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein can be subsequently identified via examining the sites to which the compounds bind. These sites can be ligand binding sites and can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the factor the complexed ligand is found.

The three dimensional geometric structure of a site, for example that of a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, can be determined by known methods in the art, such as X-ray crystallography, which can determine a complete molecular structure. Solid or liquid phase NMR can be used to determine certain intramolecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures. The geometric structures can be measured with a complexed ligand, natural or artificial, which can increase the accuracy of the active site structure determined.

Other methods for preparing or identifying peptides that bind to a target are known in the art. Molecular imprinting, for instance, can be used for the de novo construction of macromolecular structures such as peptides that bind to a molecule. See, for example, Kenneth J. Shea, Molecular Imprinting of Synthetic Network Polymers: The De Novo synthesis of Macromolecular Binding and Catalytic Sites, TRIP Vol. 2, No. 5, May 1994; Mosbach, (1994) Trends in Biochem. Sci., 19(9); and Wulff, G., in Polymeric Reagents and Catalysts (Ford, W. T., Ed.) ACS Symposium Series No. 308, pp 186-230, American Chemical Society (1986). One method for preparing mimics of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compound involves the steps of: (i) polymerization of functional monomers around a known substrate (the template) that exhibits a desired activity; (ii) removal of the template molecule; and then (iii) polymerization of a second class of monomers in, the void left by the template, to provide a new molecule which exhibits one or more desired properties which are similar to that of the template. In addition to preparing peptides in this manner other binding molecules such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and other biologically active materials can also be prepared. This method is useful for designing a wide variety of biological mimics that are more stable than their natural counterparts, because they are prepared by the free radical polymerization of functional monomers, resulting in a compound with a nonbiodegradable backbone. Other methods for designing such molecules include for example drug design based on structure activity relationships, which require the synthesis and evaluation of a number of compounds and molecular modeling.

Screening Assays

Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 Modulating Compounds. A Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compound can be a compound that affects the activity and/or expression of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein in vivo and/or in vitro. Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds can be agonists and antagonists of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, and can be compounds that exert their effect on the activity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein via the expression, via post-translational modifications, or by other means.

Test compounds or agents which bind to a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, and/or have a stimulatory or inhibitory effect on the activity or the expression of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, can be identified by two types of assays: (a) cell-based assays which utilize cells expressing a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein or a variant thereof on the cell surface; or (b) cell-free assays, which can make use of isolated Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 proteins. These assays can employ a biologically active fragment of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, full-length proteins, or a fusion protein which includes all or a portion of a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. A Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein can be obtained from any suitable mammalian species (e.g., human, rat, chick, xenopus, equine, bovine or murine). The assay can be a binding assay comprising direct or indirect measurement of the binding of a test compound. The assay can also be an activity assay comprising direct or indirect measurement of the activity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein. The assay can also be an expression assay comprising direct or indirect measurement of the expression of Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 mRNA nucleic acid sequences or a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. The various screening assays can be combined with an in vivo assay comprising measuring the effect of the test compound on the symptoms of a hair loss disorder or disease in a subject (for example, androgenetic alopecia, alopecia areata, alopecia totalis, or alopecia universalis), loss of hair pigmentation in a subject, or even hypotrichosis.

An in vivo assay can also comprise assessing the effect of a test compound on regulating hair growth in known mammalian models that display defective or aberrant hair growth phenotypes or mammals that contain mutations in the open reading frame (ORF) of nucleic acid sequences comprising a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene that affects hair growth regulation or hair density, or hair pigmentation. In one embodiment, controlling hair growth can comprise an induction of hair growth or density in the subject. Here, the compound's effect in regulating hair growth can be observed either visually via examining the organism's physical hair growth or loss, or by assessing protein or mRNA expression using methods known in the art.

Assays for screening test compounds that bind to or modulate the activity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein can also be carried out. The test compound can be obtained by any suitable means, such as from conventional compound libraries. Determining the ability of the test compound to bind to a membrane-bound form of the Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein can be accomplished via coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the cell expressing a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein can be measured by detecting the labeled compound in a complex. For example, the test compound can be labeled with ³H, ¹⁴C, ³⁵S, or ¹²⁵I, either directly or indirectly, and the radioisotope can be subsequently detected by direct counting of radioemmission or by scintillation counting. Alternatively, the test compound can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

Cell-based assays can comprise contacting a cell expressing Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 with a test agent and determining the ability of the test agent to modulate (such as increase or decrease) the activity or the expression of the membrane-bound molecule. Determining the ability of the test agent to modulate the activity of the membrane-bound Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 molecule can be accomplished by any method suitable for measuring the activity of such a molecule, such as monitoring downstream signaling events (e.g., You et al., Ann N Y Acad Sci. 2008 December; 1150:300-10; Posadas et al., Expert Rev Clin Immunol. 2009 January; 5(1):9-17; Korhonen et al., Basic Clin Pharmacol Toxicol. 2009 April; 104(4):276-84; Vital et al., Ther Clin Risk Manag. 2006 December; 2(4):365-75; Malek and Castro, Immunity. 2010 Aug. 27; 33(2):153-65; Cheng et al., Immunol Rev. 2011 May; 241(1):63-76; Lanier, Nat Immunol. 2008 May; 9(5):495-502; Lowell, Cold Spring Harb Perspect Biol. 2011 Mar. 1; 3(3). pii: a002352; Mócsai et al., Nat Rev Immunol. 2010 June; 10(6):387-402; Bradshaw, Cell Signal. 2010 August; 22(8):1175-84; Ivanenkov et al., Mini Rev Med Chem. 2011 January; 11(1):55-78; Himpe et al., Biofactors. 2009 January-February; 35(1):76-81, each of which are incorporated by reference in their entireties).

A Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein or the target of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein can be immobilized to facilitate the separation of complexed from uncomplexed forms of one or both of the proteins. Binding of a test compound to a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein or a variant thereof, or interaction of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein with a target molecule in the presence and absence of a test compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix (for example, glutathione-S-transferase (GST) fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtiter plates).

A Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, or a variant thereof, can also be immobilized via being bound to a solid support. Non-limiting examples of suitable solid supports include glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach a polypeptide (or polynucleotide) corresponding to Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 or a variant thereof, or test compound to a solid support, including use of covalent and non-covalent linkages, or passive absorption.

The expression of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein can also be monitored. For example, regulators of the expression of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein can be identified via contacting a cell with a test compound and determining the expression of a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 mRNA nucleic acid sequences in the cell. The expression level of a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 mRNA nucleic acid sequences in the cell in the presence of the test compound is compared to the protein or mRNA expression level in the absence of the test compound. The test compound can then be identified as a regulator of the expression of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein based on this comparison. For example, when expression of a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 mRNA nucleic acid sequences in the cell is statistically or significantly greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator/enhancer of expression of a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 mRNA nucleic acid sequences in the cell. The test compound can be said to be a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compound (such as an agonist).

Alternatively, when expression of a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 mRNA nucleic acid sequences in the cell is statistically or significantly less in the presence of the test compound than in its absence, the compound is identified as an inhibitor of the expression of a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 mRNA nucleic acid sequences in the cell. The test compound can also be said to be a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compound (such as an antagonist). The expression level of a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 mRNA nucleic acid sequences in the cell in cells can be determined by methods previously described.

For binding assays, the test compound can be a small molecule which binds to and occupies the binding site of a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, or a variant thereof. This can make the ligand binding site inaccessible to substrate such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules. In binding assays, either the test compound or a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label (for example, alkaline phosphatase, horseradish peroxidase, or luciferase). Detection of a test compound which is bound to a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene can then be determined via direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.

Determining the ability of a test compound to bind to a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein also can be accomplished using real-time Biamolecular Interaction Analysis (BIA) [McConnell et al., 1992, Science 257, 1906-1912; Sjolander, Urbaniczky, 1991, Anal. Chem. 63, 2338-2345]. BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (for example, BIACORE™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

To identify other proteins which bind to or interact with a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein and modulate its activity, a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene can be used as a bait protein in a two-hybrid assay or three-hybrid assay (Szabo et al., 1995, Curr. Opin. Struct. Biol. 5, 699-705; U.S. Pat. No. 5,283,317), according to methods practiced in the art. The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.

Functional Assays. Test compounds can be tested for the ability to increase or decrease the activity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, or a variant thereof. Activity can be measured after contacting a purified Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, a cell membrane preparation, or an intact cell with a test compound. A test compound that decreases the activity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95% or 100% is identified as a potential agent for decreasing the activity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, for example an antagonist. A test compound that increases the activity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95% or 100% is identified as a potential agent for increasing the activity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, for example an agonist.

Treatment and Prevention

The invention also provides a method for treating or preventing a hair-loss disorder in a subject. In one embodiment, the method comprises detecting the presence of an alteration in a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene in a sample from the subject, the presence of the alteration being indicative of a hair-loss disorder, or the predisposition to a hair-loss disorder, and, administering to the subject in need a therapeutic treatment against a hair-loss disorder. The therapeutic treatment can be a drug administration (for example, a pharmaceutical composition comprising a siRNA directed to a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 nucleic acid). In one embodiment, the therapeutic molecule to be administered comprises a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, comprising at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% of the amino acid sequence of the corresponding sequence disclosed herein, and exhibits the function of decreasing expression of a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. This can restore the capacity to initiate hair growth in cells derived from hair follicles or skin. In another embodiment, the therapeutic molecule to be administered comprises a nucleic acid sequence comprising a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene that encodes a polypeptide, comprising at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% of the nucleic acid sequence of the corresponding sequence disclosed herein, and encodes a polypeptide with the function of decreasing expression of a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, thus restoring the capacity to initiate hair growth in cells derived from hair follicle cells or skin.

The alteration can be determined at the level of the DNA, RNA, or polypeptide. Optionally, detection can be determined by performing an oligonucleotide ligation assay, a confirmation based assay, a hybridization assay, a sequencing assay, an allele-specific amplification assay, a microsequencing assay, a melting curve analysis, a denaturing high performance liquid chromatography (DHPLC) assay (for example, see Jones et al, (2000) Hum Genet., 106(6):663-8), or a combination thereof. In another embodiment, the detection is performed by sequencing all or part of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or by selective hybridization or amplification of all or part of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. A Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene specific amplification can be carried out before the alteration identification step.

An alteration in a chromosome region occupied by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene can be any form of mutation(s), deletion(s), rearrangement(s) and/or insertions in the coding and/or non-coding region of the locus, alone or in various combination(s). Mutations can include point mutations. Insertions can encompass the addition of one or several residues in a coding or non-coding portion of the gene locus. Insertions can comprise an addition of between 1 and 50 base pairs in the gene locus. Deletions can encompass any region of one, two or more residues in a coding or non-coding portion of the gene locus, such as from two residues up to the entire gene or locus. Deletions can affect smaller regions, such as domains (introns) or repeated sequences or fragments of less than about 50 consecutive base pairs, although larger deletions can occur as well. Rearrangement includes inversion of sequences. The alteration in a chromosome region occupied by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene can result in amino acid substitutions, RNA splicing or processing, product instability, the creation of stop codons, frame-shift mutations, and/or truncated polypeptide production. The alteration can result in the production of a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene with altered function, stability, targeting or structure. The alteration can also cause a reduction, or even an increase in protein expression. In one embodiment, the alteration in the chromosome region occupied by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene can comprise a point mutation, a deletion, or an insertion in a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or corresponding expression product. In another embodiment, the alteration can be a deletion or partial deletion of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. The alteration can be determined at the level of the DNA, RNA, or polypeptide.

In another embodiment, the method can comprise detecting the presence of altered RNA expression. Altered RNA expression includes the presence of an altered RNA sequence, the presence of an altered RNA splicing or processing, or the presence of an altered quantity of RNA. These can be detected by various techniques known in the art, including sequencing all or part of the RNA or by selective hybridization or selective amplification of all or part of the RNA. In a further embodiment, the method can comprise detecting the presence of altered expression of a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. Altered polypeptide expression includes the presence of an altered polypeptide sequence, the presence of an altered quantity of polypeptide, or the presence of an altered tissue distribution. These can be detected by various techniques known in the art, including by sequencing and/or binding to specific ligands (such as antibodies).

Various techniques known in the art can be used to detect or quantify altered gene or RNA expression or nucleic acid sequences, which include, but are not limited to, hybridization, sequencing, amplification, and/or binding to specific ligands (such as antibodies). Other suitable methods include allele-specific oligonucleotide (ASO), oligonucleotide ligation, allele-specific amplification, Southern blot (for DNAs), Northern blot (for RNAs), single-stranded conformation analysis (SSCA), PFGE, fluorescent in situ hybridization (FISH), gel migration, clamped denaturing gel electrophoresis, denaturing HLPC, melting curve analysis, heteroduplex analysis, RNase protection, chemical or enzymatic mismatch cleavage, ELISA, radio-immunoassays (RIA) and immuno-enzymatic assays (IEMA). Some of these approaches (such as SSCA and CGGE) are based on a change in electrophoretic mobility of the nucleic acids, as a result of the presence of an altered sequence. According to these techniques, the altered sequence is visualized by a shift in mobility on gels. The fragments can then be sequenced to confirm the alteration. Some other approaches are based on specific hybridization between nucleic acids from the subject and a probe specific for wild type or altered gene or RNA. The probe can be in suspension or immobilized on a substrate. The probe can be labeled to facilitate detection of hybrids. Some of these approaches are suited for assessing a polypeptide sequence or expression level, such as Northern blot, ELISA and RIA. These latter require the use of a ligand specific for the polypeptide, for example, the use of a specific antibody.

Sequencing. Sequencing can be carried out using techniques well known in the art, using automatic sequencers. The sequencing can be performed on the complete Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or on specific domains thereof, such as those known or suspected to carry deleterious mutations or other alterations.

Amplification. Amplification is based on the formation of specific hybrids between complementary nucleic acid sequences that serve to initiate nucleic acid reproduction. Amplification can be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). These techniques can be performed using commercially available reagents and protocols. Useful techniques in the art encompass real-time PCR, allele-specific PCR, or PCR-SSCP. Amplification usually requires the use of specific nucleic acid primers, to initiate the reaction. Nucleic acid primers useful for amplifying sequences from a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or locus are able to specifically hybridize with a portion of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene locus that flank a target region of the locus, wherein the target region is altered in certain subjects having a hair-loss disorder. In one embodiment, amplification can comprise using forward and reverse PCR primers comprising nucleotide sequences of the corresponding sequence disclosed herein.

The invention provides for a nucleic acid primer, wherein the primer can be complementary to and hybridize specifically to a portion of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 coding sequence (e.g., gene or RNA) altered in certain subjects having a hair-loss disorder. Primers of the invention can be specific for altered sequences in a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or RNA. By using such primers, the detection of an amplification product indicates the presence of an alteration in a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or the absence of such gene. Primers can also be used to identify single nucleotide polymorphisms (SNPs) located in or around a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene locus; SNPs can comprise a single nucleotide change, or a cluster of SNPs in and around a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. Examples of primers of this invention can be single-stranded nucleic acid molecules of about 5 to 60 nucleotides in length, or about 8 to about 25 nucleotides in length. The sequence can be derived directly from the sequence of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. Perfect complementarity is useful to ensure high specificity; however, certain mismatch can be tolerated. For example, a nucleic acid primer or a pair of nucleic acid primers as described above can be used in a method for detecting the presence of or a predisposition to a hair-loss disorder in a subject.

Amplification methods include, e.g., polymerase chain reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y., 1990 and PCR STRATEGIES, 1995, ed. Innis, Academic Press, Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu, Genomics 4:560, 1989; Landegren, Science 241:1077, 1988; Barringer, Gene 89:117, 1990); transcription amplification (see, e.g., Kwoh, Proc. Natl. Acad. Sci. USA 86:1173, 1989); and, self-sustained sequence replication (see, e.g., Guatelli, Proc. Natl. Acad. Sci. USA 87:1874, 1990); Q Beta replicase amplification (see, e.g., Smith, J. Clin. Microbiol. 35:1477-1491, 1997), automated Q-beta replicase amplification assay (see, e.g., Burg, Mol. Cell. Probes 10:257-271, 1996) and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); see also Berger, Methods Enzymol. 152:307-316, 1987; Sambrook; Ausubel; U.S. Pat. Nos. 4,683,195 and 4,683,202; Sooknanan, Biotechnology 13:563-564, 1995. All the references stated above, an throughout the description, are incorporated by reference in their entireties.

Selective Hybridization. Hybridization detection methods are based on the formation of specific hybrids between complementary nucleic acid sequences that serve to detect nucleic acid sequence alteration(s). A detection technique involves the use of a nucleic acid probe specific for wild type or altered gene or RNA, followed by the detection of the presence of a hybrid. The probe can be in suspension or immobilized on a substrate or support (for example, as in nucleic acid array or chips technologies). The probe can be labeled to facilitate detection of hybrids. For example, a sample from the subject can be contacted with a nucleic acid probe specific for a wild type Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or an altered Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, and the formation of a hybrid can be subsequently assessed. In one embodiment, the method comprises contacting simultaneously the sample with a set of probes that are specific, respectively, for a wild type Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene and for various altered forms thereof. Thus, it is possible to detect directly the presence of various forms of alterations in a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene in the sample. Also, various samples from various subjects can be treated in parallel.

According to the invention, a probe can be a polynucleotide sequence which is complementary to and can specifically hybridize with a (target portion of a) Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or RNA, and that is suitable for detecting polynucleotide polymorphisms associated with alleles of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene (or genes) which predispose to or are associated with a hair-loss disorder. Useful probes are those that are complementary to a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, RNA, or target portion thereof. Probes can comprise single-stranded nucleic acids of between 8 to 1000 nucleotides in length, for instance between 10 and 800, between 15 and 700, or between 20 and 500. Longer probes can be used as well. A useful probe of the invention is a single stranded nucleic acid molecule of between 8 to 500 nucleotides in length, which can specifically hybridize to a region of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or RNA that carries an alteration. For example, the probe can be directed to a chromosome region occupied by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene.

The sequence of the probes can be derived from the sequences of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene and RNA as provided herein. Nucleotide substitutions can be performed, as well as chemical modifications of the probe. Such chemical modifications can be accomplished to increase the stability of hybrids (e.g., intercalating groups) or to label the probe. Some examples of labels include, without limitation, radioactivity, fluorescence, luminescence, and enzymatic labeling.

A guide to the hybridization of nucleic acids is found in e.g., Sambrook, ed., Molecular Cloning: A Laboratory Manual (3^(rd) Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, 2001; Current Protocols in Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc., New York, 1997; Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization with Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y., 1993.

Specific Ligand Binding

As discussed herein, alteration in a chromosome region occupied by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or alteration in expression of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, can also be detected by screening for alteration(s) in a sequence or expression level of a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. Different types of ligands can be used, such as specific antibodies. In one embodiment, the sample is contacted with an antibody specific for a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene and the formation of an immune complex is subsequently determined. Various methods for detecting an immune complex can be used, such as ELISA, radioimmunoassays (RIA) and immuno-enzymatic assays (IEMA).

For example, an antibody can be a polyclonal antibody, a monoclonal antibody, as well as fragments or derivatives thereof having substantially the same antigen specificity. Fragments include Fab, Fab′2, or CDR regions. Derivatives include single-chain antibodies, humanized antibodies, or poly-functional antibodies. An antibody specific for a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene can be an antibody that selectively binds such a polypeptide, namely, an antibody raised against a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or an epitope-containing fragment thereof. Although non-specific binding towards other antigens can occur, binding to the target polypeptide occurs with a higher affinity and can be reliably discriminated from non-specific binding. In one embodiment, the method can comprise contacting a sample from the subject with an antibody specific for a wild type or an altered form of a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, and determining the presence of an immune complex. Optionally, the sample can be contacted to a support coated with antibody specific for the wild type or altered form of a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. In one embodiment, the sample can be contacted simultaneously, or in parallel, or sequentially, with various antibodies specific for different forms of a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, such as a wild type and various altered forms thereof.

Gene Therapy and Protein Replacement Methods

Delivery of nucleic acids into viable cells can be effected ex vivo, in situ, or in vivo by use of vectors, such as viral vectors (e.g., lentivirus, adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). Non-limiting techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, and the calcium phosphate precipitation method (See, for example, Anderson, Nature, supplement to vol. 392, no. 6679, pp. 25-20 (1998)). Introduction of a nucleic acid or a gene encoding a polypeptide of the invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). Cells can also be cultured ex vivo in the presence of therapeutic compositions of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.

Nucleic acids can be inserted into vectors and used as gene therapy vectors. A number of viruses have been used as gene transfer vectors, including papovaviruses, e.g., SV40 (Madzak et al., 1992), adenovirus (Berkner, 1992; Berkner et al., 1988; Gorziglia and Kapikian, 1992; Quantin et al., 1992; Rosenfeld et al., 1992; Wilkinson et al., 1992; Stratford-Perricaudet et al., 1990), vaccinia virus (Moss, 1992), adeno-associated virus (Muzyczka, 1992; Ohi et al., 1990), herpesviruses including HSV and EBV (Margolskee, 1992; Johnson et al., 1992; Fink et al., 1992; Breakfield and Geller, 1987; Freese et al., 1990), and retroviruses of avian (Biandyopadhyay and Temin, 1984; Petropoulos et al., 1992), murine (Miller, 1992; Miller et al., 1985; Sorge et al., 1984; Mann and Baltimore, 1985; Miller et al., 1988), and human origin (Shimada et al., 1991; Helseth et al., 1990; Page et al., 1990; Buchschacher and Panganiban, 1992). Non-limiting examples of in vivo gene transfer techniques include transfection with viral (e.g., retroviral) vectors (see U.S. Pat. No. 5,252,479, which is incorporated by reference in its entirety) and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11:205-210 (1993), incorporated entirely by reference). For example, naked DNA vaccines are generally known in the art; see Brower, Nature Biotechnology, 16:1304-1305 (1998), which is incorporated by reference in its entirety. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Pat. No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

For reviews of gene therapy protocols and methods see Anderson et al., Science 256:808-813 (1992); U.S. Pat. Nos. 5,252,479, 5,747,469, 6,017,524, 6,143,290, 6,410,010 6,511,847; and U.S. Application Publication Nos. 2002/0077313 and 2002/00069, which are all hereby incorporated by reference in their entireties. For additional reviews of gene therapy technology, see Friedmann, Science, 244:1275-1281 (1989); Verma, Scientific American: 68-84 (1990); Miller, Nature, 357: 455-460 (1992); Kikuchi et al., J Dermatol Sci. 2008 May; 50(2):87-98; Isaka et al., Expert Opin Drug Deliv. 2007 September; 4(5):561-71; Jager et al., Curr Gene Ther. 2007 August; 7(4):272-83; Waehler et al., Nat Rev Genet. 2007 August; 8(8):573-87; Jensen et al., Ann Med. 2007; 39(2):108-15; Herweijer et al., Gene Ther. 2007 January; 14(2):99-107; Eliyahu et al., Molecules, 2005 Jan. 31; 10(1):34-64; and Altaras et al., Adv Biochem Eng Biotechnol. 2005; 99:193-260, all of which are hereby incorporated by reference in their entireties.

Protein replacement therapy can increase the amount of protein by exogenously introducing wild-type or biologically functional protein by way of infusion. A replacement polypeptide can be synthesized according to known chemical techniques or can be produced and purified via known molecular biological techniques. Protein replacement therapy has been developed for various disorders. For example, a wild-type protein can be purified from a recombinant cellular expression system (e.g., mammalian cells or insect cells-see U.S. Pat. No. 5,580,757 to Desnick et al.; U.S. Pat. Nos. 6,395,884 and 6,458,574 to Selden et al.; U.S. Pat. No. 6,461,609 to Calhoun et al.; U.S. Pat. No. 6,210,666 to Miyamura et al.; U.S. Pat. No. 6,083,725 to Selden et al.; U.S. Pat. No. 6,451,600 to Rasmussen et al.; U.S. Pat. No. 5,236,838 to Rasmussen et al. and U.S. Pat. No. 5,879,680 to Ginns et al.), human placenta, or animal milk (see U.S. Pat. No. 6,188,045 to Reuser et al.), or other sources known in the art. After the infusion, the exogenous protein can be taken up by tissues through non-specific or receptor-mediated mechanism.

A polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene can also be delivered in a controlled release system. For example, the polypeptide can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump can be used (see is Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

Pharmaceutical Compositions and Administration for Therapy

Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 proteins and Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds of the invention can be administered to the subject once (e.g., as a single injection or deposition). Alternatively, Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 proteins and Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds can be administered once or twice daily to a subject in need thereof for a period of from about two to about twenty-eight days, or from about seven to about ten days. Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 proteins and Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds can also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof. Furthermore, Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 proteins and Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds of the invention can be co-administrated with another therapeutic. Where a dosage regimen comprises multiple administrations, the effective amount of the Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 proteins and Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds administered to the subject can comprise the total amount of gene product administered over the entire dosage regimen.

Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 proteins and Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds can be administered to a subject by any means suitable for delivering the Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 proteins and Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds to cells of the subject, such as the dermis, epidermis, dermal papilla cells, or hair follicle cells. For example, Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 proteins and Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds can be administered by methods suitable to transfect cells. Transfection methods for eukaryotic cells are well known in the art, and include direct injection of the nucleic acid into the nucleus or pronucleus of a cell; electroporation; liposome transfer or transfer mediated by lipophilic materials; receptor mediated nucleic acid delivery, bioballistic or particle acceleration; calcium phosphate precipitation, and transfection mediated by viral vectors.

The compositions of this invention can be formulated and administered to reduce the symptoms associated with a hair-loss disorder by any means that produces contact of the active ingredient with the agent's site of action in the body of a subject, such as a human or animal (e.g., a dog, cat, or horse). They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

A therapeutically effective dose of Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds can depend upon a number of factors known to those or ordinary skill in the art. The dose(s) of the Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds can vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds to have upon the nucleic acid or polypeptide of the invention. These amounts can be readily determined by a skilled artisan. Any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human.

Pharmaceutical compositions for use in accordance with the invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The therapeutic compositions of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration. Techniques and formulations generally can be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. (20^(th) Ed., 2000), the entire disclosure of which is herein incorporated by reference. For systemic administration, an injection is useful, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the therapeutic compositions of the invention can be formulated in liquid solutions, for example in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the therapeutic compositions can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. These pharmaceutical formulations include formulations for human and veterinary use.

According to the invention, a pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active compound can be used. Supplementary active compounds can also be incorporated into the compositions.

The invention also provides for a kit that comprises a pharmaceutically acceptable carrier and a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compound identified using the screening assays of the invention packaged with instructions for use. For modulators that are antagonists of the activity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, or which reduce the expression of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, the instructions would specify use of the pharmaceutical composition for promoting the loss of hair on the body surface of a mammal (for example, arms, legs, bikini area, face).

For Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds that are agonists of the activity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein or increase the expression of one or more proteins encoded by Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 genes, the instructions would specify use of the pharmaceutical composition for regulating hair growth. In one embodiment, the instructions would specify use of the pharmaceutical composition for the treatment of hair loss disorders. In a further embodiment, the instructions would specify use of the pharmaceutical composition for restoring hair pigmentation. For example, administering an agonist can reduce hair graying in a subject.

A pharmaceutical composition containing a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compound can be administered in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed herein. Such pharmaceutical compositions can comprise, for example antibodies directed to polypeptides encoded by genes comprising a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, or variants thereof, or agonists and antagonists of a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. The compositions can be administered alone or in combination with at least one other agent, such as a stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.

Sterile injectable solutions can be prepared by incorporating the Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In some embodiments, the Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compound can be applied via transdermal delivery systems, which slowly releases the active compound for percutaneous absorption. Permeation enhancers can be used to facilitate transdermal penetration of the active factors in the conditioned media. Transdermal patches are described in for example, U.S. Pat. Nos. 5,407,713; 5,352,456; 5,332,213; 5,336,168; 5,290,561; 5,254,346; 5,164,189; 5,163,899; 5,088,977; 5,087,240; 5,008,110; and 4,921,475.

Various routes of administration and various sites of cell implantation can be utilized, such as, subcutaneous or intramuscular, in order to introduce the aggregated population of cells into a site of preference. Once implanted in a subject (such as a mouse, rat, or human), the aggregated cells can then stimulate the formation of a hair follicle and the subsequent growth of a hair structure at the site of introduction. In another embodiment, transfected cells (for example, cells expressing a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene are implanted in a subject to promote the formation of hair follicles within the subject. In further embodiments, the transfected cells are cells derived from the end bulb of a hair follicle (such as dermal papilla cells or dermal sheath cells). Aggregated cells (for example, cells grown in a hanging drop culture) or transfected cells (for example, cells produced as described herein) maintained for 1 or more passages can be introduced (or implanted) into a subject (such as a rat, mouse, dog, cat, human, and the like).

“Subcutaneous” administration can refer to administration just beneath the skin (i.e., beneath the dermis). Generally, the subcutaneous tissue is a layer of fat and connective tissue that houses larger blood vessels and nerves. The size of this layer varies throughout the body and from person to person. The interface between the subcutaneous and muscle layers can be encompassed by subcutaneous administration.

This mode of administration can be feasible where the subcutaneous layer is sufficiently thin so that the factors present in the compositions can migrate or diffuse from the locus of administration and contact the hair follicle cells responsible for hair formation. Thus, where intradermal administration is utilized, the bolus of composition administered is localized proximate to the subcutaneous layer.

Administration of the cell aggregates (such as DP or DS aggregates) is not restricted to a single route, but can encompass administration by multiple routes. For instance, exemplary administrations by multiple routes include, among others, a combination of intradermal and intramuscular administration, or intradermal and subcutaneous administration. Multiple administrations can be sequential or concurrent. Other modes of application by multiple routes will be apparent to the skilled artisan.

In other embodiments, this implantation method will be a one-time treatment for some subjects. In further embodiments of the invention, multiple cell therapy implantations will be required. In some embodiments, the cells used for implantation will generally be subject-specific genetically engineered cells. In another embodiment, cells obtained from a different species or another individual of the same species can be used. Thus, using such cells can require administering an immunosuppressant to prevent rejection of the implanted cells. Such methods have also been described in United States Patent Application Publication 2004/0057937 and PCT application publication WO 2001/32840, and are hereby incorporated by reference.

Inhibitors

Cytokines are produced to activate neighboring cells to communicate danger signals to one another and spread and amplify the inflammatory response. Over the years, it was learned how to both neutralize these cytokines with blocking antibodies and inhibit their signaling in responding cells by small molecule protein tyrosine kinase inhibitors FDA approved drugs exist for both approaches, e.g., IL-2 and TNF blocking antibodies and the small orally available molecule Gleevec™ that blocks cytokine signaling. Whenever possible, the center will pursue topical small molecule formulations that should improve efficacy while limiting systemic toxicity (improved therapeutic indexes) encouraging clinical evaluation in AA of other small molecule inhibitors in the biopharmaceutical pipeline.

To block signaling of cytokine receptors, topical/oral JAK1/2 inhibitor (Incyte), which has already demonstrated safety and efficacy in patients with psoriaisis and RA, can be used. Similarly, topical and/or oral JAK3 inhibitors as described herein (e.g., tofacitinib (CP690550), R348 and VX-509), which have demonstrated preliminary safety and efficacy in patients with RA, can be used.

The inhibitors can comprise peptides (such as antibodies or fragments thereof), small molecules, nucleic acids (such as siRNA or antisense RNA), or other agents) that can bind to a polypeptide molecule encoded by a gene of interest and/or molecules that have an inhibitory effect on the biological activity of a protein of interest or its expression.

As used herein, a “Jak 1 inhibitor” refers to a compound that interacts with a Jak 1 gene or a Jak 1 protein or polypeptide and inhibits its activity and/or its expression. The compound can decrease the activity or expression of a protein encoded by Jak 1.

In one embodiment, a Jak 1 inhibitor can be a peptide fragment that binds a protein comprising SEQ ID NO: 1. For example, the fragment can encompass any portion of at least about 8 consecutive amino acids of SEQ ID NO: 1. The fragment can comprise at least about 10 consecutive amino acids, at least about 20 consecutive amino acids, at least about 30 consecutive amino acids, at least about 40 consecutive amino acids, at least about 50 consecutive amino acids, at least about 60 consecutive amino acids, or at least about 75 consecutive amino acids of SEQ ID NO: 1. Fragments include all possible amino acid lengths between and including about 8 and about 100 amino acids, for example, lengths between about 10 and about 100 amino acids, between about 15 and about 100 amino acids, between about 20 and about 100 amino acids, between about 35 and about 100 amino acids, between about 40 and about 100 amino acids, between about 50 and about 100 amino acids, between about 70 and about 100 amino acids, between about 75 and about 100 amino acids, or between about 80 and about 100 amino acids. These peptide fragments can be obtained commercially or synthesized via liquid phase or solid phase synthesis methods (Atherton et al., (1989) Solid Phase Peptide Synthesis: a Practical Approach. TRL Press, Oxford, England).

As used herein, a “Jak 2 inhibitor” refers to a compound that interacts with a Jak 2 gene or a Jak 2 protein or polypeptide and inhibits its activity and/or its expression. The compound can decrease the activity or expression of a protein encoded by Jak 2.

In one embodiment, a Jak 2 inhibitor can be a peptide fragment that binds a protein comprising SEQ ID NO: 3. For example, the fragment can encompass any portion of at least about 8 consecutive amino acids of SEQ ID NO: 3. The fragment can comprise at least about 10 consecutive amino acids, at least about 20 consecutive amino acids, at least about 30 consecutive amino acids, at least about 40 consecutive amino acids, at least about 50 consecutive amino acids, at least about 60 consecutive amino acids, or at least about 75 consecutive amino acids of SEQ ID NO: 3. Fragments include all possible amino acid lengths between and including about 8 and about 100 amino acids, for example, lengths between about 10 and about 100 amino acids, between about 15 and about 100 amino acids, between about 20 and about 100 amino acids, between about 35 and about 100 amino acids, between about 40 and about 100 amino acids, between about 50 and about 100 amino acids, between about 70 and about 100 amino acids, between about 75 and about 100 amino acids, or between about 80 and about 100 amino acids. These peptide fragments can be obtained commercially or synthesized via liquid phase or solid phase synthesis methods (Atherton et al., (1989) Solid Phase Peptide Synthesis: a Practical Approach. IRL Press, Oxford, England).

As used herein, a “Jak 3 inhibitor” can be a compound that interacts with a Jak 3 gene, or a Jak 3 protein or polypeptide, and inhibits its activity and/or its expression. The compound can decrease the activity or expression of a protein encoded by Jak 3. In one embodiment, a Jak3 inhibitor can be a Jak3 modulating compound.

In one embodiment, a Jak 3 inhibitor can be a peptide fragment that binds a protein comprising SEQ ID NO: 109. For example, the fragment can encompass any portion of about 8 consecutive amino acids of SEQ ID NO: 109. The fragment can comprise about 10 consecutive amino acids, about 20 consecutive amino acids, about 30 consecutive amino acids, about 40 consecutive amino acids, about 50 consecutive amino acids, about 60 consecutive amino acids, or about 75 consecutive amino acids of SEQ ID NO: 109. Fragments include all possible amino acid lengths between and including about 8 and about 100 amino acids, for example, lengths between about 10 and about 100 amino acids, between about 15 and about 100 amino acids, between about 20 and about 100 amino acids, between about 35 and about 100 amino acids, between about 40 and about 100 amino acids, between about 50 and about 100 amino acids, between about 70 and about 100 amino acids, between about 75 and about 100 amino acids, or between about 80 and about 100 amino acids. These peptide fragments can be obtained commercially or synthesized via liquid phase or solid phase synthesis methods (Atherton et al., (1989) Solid Phase Peptide Synthesis: a Practical Approach. IRL Press, Oxford, England).

An inhibitor of the invention can be a protein, such as an antibody (monoclonal, polyclonal, humanized, chimeric, or fully human), or a binding fragment thereof, directed against a polypeptide encoded by SEQ ID NO: 109. An antibody fragment can be a form of an antibody other than the full-length form and includes portions or components that exist within full-length antibodies, in addition to antibody fragments that have been engineered. Antibody fragments can include, but are not limited to, single chain Fv (scFv), diabodies, Fv, and (Fab′)₂, triabodies, Fc, Fab, CDR1, CDR2, CDR3, combinations of CDR's, variable regions, tetrabodies, bifunctional hybrid antibodies, framework regions, constant regions, and the like (see, Maynard et al., (2000) Ann. Rev. Biomed. Eng. 2:339-76; Hudson (1998) Curr. Opin. Biotechnol. 9:395-402). Antibodies can be obtained commercially, custom generated, or synthesized against an antigen of interest according to methods established in the art (see Roland E. Kontermann and Stefan Dübel (editors), Antibody Engineering, Vol. I & II, (2010) 2^(nd) ed., Springer; Antony S. Dimitrov (editor), Therapeutic Antibodies: Methods and Protocols (Methods in Molecular Biology), (2009), Humana Press; Benny Lo (editor) Antibody Engineering: Methods and Protocols (Methods in Molecular Biology), (2004) Humana Press, each of which are hereby incorporated by reference in their entireties).

As used herein, a “Stat 1 inhibitor” refers to a compound that interacts with a Stat 1 gene or a Stat 1 protein or polypeptide and inhibits its activity and/or its expression. The compound can decrease the activity or expression of a protein encoded by Stat 1.

In one embodiment, a Stat 1 inhibitor can be a peptide fragment that binds a protein comprising SEQ ID NO: 5. For example, the fragment can encompass any portion of at least about 8 consecutive amino acids of SEQ ID NO: 5. The fragment can comprise at least about 10 consecutive amino acids, at least about 20 consecutive amino acids, at least about 30 consecutive amino acids, at least about 40 consecutive amino acids, at least about 50 consecutive amino acids, at least about 60 consecutive amino acids, or at least about 75 consecutive amino acids of SEQ ID NO: 5. Fragments include all possible amino acid lengths between and including about 8 and about 100 amino acids, for example, lengths between about 10 and about 100 amino acids, between about 15 and about 100 amino acids, between about 20 and about 100 amino acids, between about 35 and about 100 amino acids, between about 40 and about 100 amino acids, between about 50 and about 100 amino acids, between about 70 and about 100 amino acids, between about 75 and about 100 amino acids, or between about 80 and about 100 amino acids. These peptide fragments can be obtained commercially or synthesized via liquid phase or solid phase synthesis methods (Atherton et al., (1989) Solid Phase Peptide Synthesis: a Practical Approach. IRL Press, Oxford, England).

As used herein, a “Stat 2 inhibitor” refers to a compound that interacts with a Stat 2 gene or a Stat 2 protein or polypeptide and inhibits its activity and/or its expression. The compound can decrease the activity or expression of a protein encoded by Stat 2.

In one embodiment, a Stat 2 inhibitor can be a peptide fragment that binds a protein comprising SEQ ID NO: 7. For example, the fragment can encompass any portion of at least about 8 consecutive amino acids of SEQ ID NO: 7. The fragment can comprise at least about 10 consecutive amino acids, at least about 20 consecutive amino acids, at least about 30 consecutive amino acids, at least about 40 consecutive amino acids, at least about 50 consecutive amino acids, at least about 60 consecutive amino acids, or at least about 75 consecutive amino acids of SEQ ID NO: 7. Fragments include all possible amino acid lengths between and including about 8 and about 100 amino acids, for example, lengths between about 10 and about 100 amino acids, between about 15 and about 100 amino acids, between about 20 and about 100 amino acids, between about 35 and about 100 amino acids, between about 40 and about 100 amino acids, between about 50 and about 100 amino acids, between about 70 and about 100 amino acids, between about 75 and about 100 amino acids, or between about 80 and about 100 amino acids. These peptide fragments can be obtained commercially or synthesized via liquid phase or solid phase synthesis methods (Atherton et al., (1989) Solid Phase Peptide Synthesis: a Practical Approach. IRL Press, Oxford, England).

As used herein, a “Jak/Stat inhibitor” refers to a compound that interacts with a Jak1/Jak2/Jak3/Stat1/Stat2 gene or a Jak1/Jak2/Jak3/Stat1/Stat2 protein or polypeptide and inhibits its activity and/or its expression. The compound can decrease the activity or expression of a protein encoded by Jak1/Jak2/Jak3/Stat1/Stat2.

An inhibitor of the invention can be a protein, such as an antibody (monoclonal, polyclonal, humanized, chimeric, or fully human), or a binding fragment thereof, directed against a polypeptide encoded by the corresponding sequence disclosed herein. An antibody fragment can be a form of an antibody other than the full-length form and includes portions or components that exist within full-length antibodies, in addition to antibody fragments that have been engineered. Antibody fragments can include, but are not limited to, single chain Fv (scFv), diabodies, Fv, and (Fab′)₂, triabodies, Fc, Fab, CDR1, CDR2, CDR3, combinations of CDR's, variable regions, tetrabodies, bifunctional hybrid antibodies, framework regions, constant regions, and the like (see, Maynard et al., (2000) Ann. Rev. Biomed. Eng. 2:339-76; Hudson (1998) Curr. Opin. Biotechnol. 9:395-402). Antibodies can be obtained commercially, custom generated, or synthesized against an antigen of interest according to methods established in the art (Janeway et al., (2001) Immunobiology, 5th ed., Garland Publishing).

An inhibitor of the invention can also be a small molecule that binds to a protein and disrupts its function. Small molecules are a diverse group of synthetic and natural substances generally having low molecular weights. They can be isolated from natural sources (for example, plants, fungi, microbes and the like), are obtained commercially and/or available as libraries or collections, or synthesized. Candidate small molecules that modulate a protein can be identified via in silico screening or high-through-put (HTP) screening of combinatorial libraries. Most conventional pharmaceuticals, such as aspirin, penicillin, and many chemotherapeutics, are small molecules, can be obtained commercially, can be chemically synthesized, or can be obtained from random or combinatorial libraries (Werner et al., (2006) Brief Funct. Genomic Proteomic 5(1):32-6). In some embodiments, the agent is a small molecule that binds, interacts, or associates with a target protein or RNA. Such a small molecule can be an organic molecule that, when the target is an intracellular target, is capable of penetrating the lipid bilayer of a cell to interact with the target. Small molecules include, but are not limited to, toxins, chelating agents, metals, and metalloid compounds. Small molecules can be attached or conjugated to a targeting agent so as to specifically guide the small molecule to a particular cell.

Pharmaceutical Compositions and Administration for Therapy

An inhibitor or agonist of the invention can be incorporated into pharmaceutical compositions suitable for administration, for example the inhibitor and a pharmaceutically acceptable carrier.

According to the invention, a pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active compound can be used. Supplementary active compounds can also be incorporated into the compositions.

Any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human.

A pharmaceutical composition of the invention can be administered in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed herein. Such pharmaceutical compositions can comprise, for example antibodies directed to polypeptides. The compositions can be administered alone or in combination with at least one other agent, such as a stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the inhibitor (e.g., a polypeptide or antibody or small molecule) or agonist of the invention in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The dosage administered can be a therapeutically effective amount of the composition sufficient to result in amelioration of symptoms of alopecia areata, and can vary depending upon known factors such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired.

In some embodiments, the effective amount of the administered Jak3 modulating compound (e.g., a compound described herein that is directed to Jak3, such as a small molecule JAK3 inhibitor) is about 0.0001 μg/kg body weight, about 0.00025 μg/kg body weight, about 0.0005 μg/kg body weight, about 0.00075 μg/kg body weight, about 0.001 μg/kg body weight, about 0.0025 μg/kg body weight, about 0.005 μg/kg body weight, about 0.0075 μg/kg body weight, about 0.01 μg/kg body weight, about 0.025 μg/kg body weight, about 0.05 μg/kg body weight, about 0.075 μg/kg body weight, about 0.1 μg/kg body weight, about 0.25 μg/kg body weight, about 0.5 μg/kg body weight, about 0.75 μg/kg body weight, about 1 μg/kg body weight, about 5 μg/kg body weight, about 10 μg/kg body weight, about 25 μg/kg body weight, about 50 μg/kg body weight, about 75 μg/kg body weight, about 100 μg/kg body weight, about 150 μg/kg body weight, about 200 μg/kg body weight, about 250 μg/kg body weight, about 300 μg/kg body weight, about 350 μg/kg body weight, about 400 μg/kg body weight, about 450 μg/kg body weight, about 500 μg/kg body weight, about 550 μg/kg body weight, about 600 μg/kg body weight, about 650 μg/kg body weight, about 700 μg/kg body weight, about 750 μg/kg body weight, about 800 μg/kg body weight, about 850 μg/kg body weight, about 900 μg/kg body weight, about 950 μg/kg body weight, about 1000 μg/kg body weight, about 2000 μg/kg body weight, about 3000 μg/kg body weight, about 4000 μg/kg body weight, about 5000 μg/kg body weight, about 6000 μg/kg body weight, about 7000 μg/kg body weight, about 8000 μg/kg body weight, about 95000 μg/kg body weight, or about 10,000 μg/kg body weight.

In some embodiments, the effective amount of the administered Jak3 modulating compound (e.g., a small molecule JAK3 inhibitor) is about 1 mg/kg body weight, about 1.5 mg/kg body weight, about 2 mg/kg body weight, about 2.5 mg/kg body weight, about 3 mg/kg body weight, about 3.5 mg/kg body weight, about 4 mg/kg body weight, about 4.5 mg/kg body weight, about 5 mg/kg body weight, about 5.5 mg/kg body weight, about 6 mg/kg body weight, about 6.5 mg/kg body weight, about 7 mg/kg body weight, about 7.5 mg/kg body weight, about 8 mg/kg body weight, about 9.5 mg/kg body weight, about 10 mg/kg body weight, about 10.5 mg/kg body weight, about 11.0 mg/kg body weight, about 11.5 mg/kg body weight, about 12 mg/kg body weight, about 12.5 mg/kg body weight, about 13 mg/kg body weight, about 13.5 mg/kg body weight, about 14 mg/kg body weight, about 14.5 mg/kg body weight, about 15 mg/kg body weight, about 15.5 mg/kg body weight, about 16 mg/kg body weight, about 16.5 mg/kg body weight, about 17 mg/kg body weight, about 17.5 mg/kg body weight, about 18 mg/kg body weight, about 19.5 mg/kg body weight, about 20 mg/kg body weight, about 21.5 mg/kg body weight, about 22 mg/kg body weight, about 22.5 mg/kg body weight, about 23 mg/kg body weight, about 23.5 mg/kg body weight, about 24 mg/kg body weight, about 24.5 mg/kg body weight, about 25 mg/kg body weight, about 25.5 mg/kg body weight, about 26 mg/kg body weight, about 26.5 mg/kg body weight, about 27 mg/kg body weight, about 27.5 mg/kg body weight, about 28 mg/kg body weight, about 29.5 mg/kg body weight, or about 30 mg/kg body weight.

In other embodiments, the administered Jak3 modulating compound (e.g., a compound described herein that is directed to Jak3, such as a small molecule JAK3 inhibitor), can be administered as a topical cream. In some embodiments, the effective amount of the administered Jak3 modulating compound (such as a small molecule JAK3 inhibitor) is present at a concentration of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3.0%. In some embodiments, the effective amount of the administered Jak3 modulating compound (such as a small molecule JAK3 inhibitor) is present at a concentration of about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or of about 10%.

In some embodiments, the Jak3 modulating compound (e.g., a compound described herein that is directed to Jak3, such as a small molecule JAK3 inhibitor), can be co-administered with a second JAK inhibitor (such as a JAK1/2 inhibitor). In one embodiment, the JAK1/2 inhibitor can be administered as a topical cream. In some embodiments, the effective amount of the administered small molecule JAK1/2 inhibitor is present at a concentration of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3.0%. In some embodiments, the effective amount of the administered JAK1/2 inhibitor is present at a concentration of about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or of about 10%.

Toxicity and therapeutic efficacy of therapeutic compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Therapeutic agents that exhibit large therapeutic indices are useful. Therapeutic compositions that exhibit some toxic side effects can be used.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated by reference. Publications and references cited herein are not admitted to be prior art.

EXAMPLES

Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.

Example 1

Interferon γ has been shown to be a target in murine AA (see Nakamura, et al. 2008, Am J Pathol, 172(3): 650-658; see also Freyschmidt-Paul et al., Br J Dermatol., 155(3):515-521; see also Gilhar et al., Journal Invest. Dermatol., 124(1):288-289). In human AA, AA PBMCs show Th1 skewing, AA skin shows IFN signature and GWAS genes (SOCS/IFN-g/). but also IL-2/6/13/21/26.

The interferon γ pathway (FIG. 7 ) induces NKG2DL in target HF cells, enhances antigen presentation by DCs, promotes Th1 cellular autoimmunity and augments NK and CTL mediated-cytolysis.

JAK1/2 inhibitors in clinical development include a) INCB018424, topical and oral; 5 nM activity (Incyte); b) CEP-701 (Cephalon); and c) TG101348.

TABLE 4 INCB18424-203: Treatment-Emergent Adverse Events in Descending Order (all casualties) (oral delivery is considered an acceptable profile) Vehicle Active Medra Preferred Term (n = 50) (n = 149) Application site irritation, pruritus 7 (14.0) 14 (9.3) Nasopharyngitis 1 (2.0) 10 (6.7) Upper respiratory tract infection 3 (6.0) 10 (6.7) Sinusitis 1 (2.0) 6 (4.0) Back Pain 0 (0.0) 5 (3.4) Headache 2 (0.0) 4 (2.7) Influenza 2 (4.0) 4 (2.7) Abdominal pain 0 (0.0) 3 (2.0) Six SAEs, none treatment related: prostate ca, prolonged hospitalization for cholecystitis, cholelithiasis (1.5%); herniated disc repair, coronary occlusion₁₆ with hx ASHD (0.5%). CVA (vehicle)

Clinical programs in AA include the following:

-   Mild-Moderate AA: Phase 2/3 Psoriasis and oral Phase 3     Myelofibrosis, using a topical JAK inhibitor

A population-based cohort evaluated cases ascertained through National Alopecia Areata Registry vs. controls ascertained through New York City Cancer project. They were matched for Northern European ancestry using AIMS.

-   Cases: n=1080 (Alopecia Universalis=482; Alopecia Totalis=120;     Alopecia Areata Transient=176; Alopecia Areata Patchy=302) -   Controls: n=1053

FIG. 9 presents a Genomewide Association Study (GWAS) in alopecia areata.

TABLE 5 List of Genes implicated in AA. Stongest association Maximum Region Gene Function (pvalue) odds ratio 2q33.2 CTLA4 T-cell proliferation 3.55 × 10⁻¹³ 1.44 ICOS T-cell proliferation 4.33 × 10⁻⁰⁸ 1.32 4q27 IL21/IL2 T-, B- and NK-cell 4.27 × 10⁻⁰⁸ 1.34 proliferation 6q25.1 ULBP6 NKG2D activating 4.49 × 10⁻¹⁹ 1.65 ligand ULBP3 NKG2D activating 4.43 × 10⁻¹⁷ 1.52 ligand 9q31.1 STX17 premature hair graying 3.60 × 10⁻⁰⁷ 1.33 10p15.1 IL2RA T-cell proliferation 1.74 × 10⁻¹² 1.41 (CD25) 11q13 PRDX5 antioxidant enzyme 4.14 × 10⁻⁰⁷ 1.33 12q13 Eos T-cell proliferation 3.21 × 10⁻⁰⁸ 1.34 (IKZF4) ERBB3 epidermal growth 1.27 × 10⁻⁰⁷ 1.34 factor receptor 6p21.32 MICA NKG2D activating 1.19 × 10⁻⁰⁷ 1.44 (HLA) ligand NOTCH4 T-cell differentiation 1.03 × 10⁻⁰⁸ 1.61 BTNL2 T-cell differentiation 2.11 × 10⁻²⁶ 2.70 HLA-DRA Antigen presentation 2.93 × 10⁻³¹ 2.62 HLA-DQA1 Antigen presentation 3.60 × 10⁻¹⁷ 2.15 HLA-DQA2 Antigen presentation 1.38 × 10⁻³⁵ 5.43 HLA-DQB2 Antigen presentation 1.73 × 10⁻¹³ 1.60 HLA-DOB Antigen presentation 2.07 × 10⁻⁰⁸ 1.72

TABLE 6 Common Cause Model of Skin Autoimmune Diseases. See also Jin et al., 2010, NEJM, 362: 1686-1697. Stongest association Maximum Region Gene (pvalue) odds ratio Involved in other autoimmune disease 2q33.2 CTLA4 3.55 × 10⁻¹³ 1.44 T1D, RA, CeD, MS, SLE, GD ICOS 4.33 × 10⁻⁰⁸ 1.32 T1D, RA, CeD, MS, SLE, GD 4q27 IL21/IL2 4.27 × 10⁻⁰⁸ 1.34 ● T1D, RA, CeD, PS 6q25.1 ULBP6 4.49 × 10⁻¹⁹ 1.65 none ULBP3 4.43 × 10⁻¹⁷ 1.52 none 9q31.1 STX17 3.60 × 10⁻⁰⁷ 1.33 none 10p15.1 IL2RA (CD25) 1.74 × 10⁻¹² 1.41 ◯ T1D, MS, GD, GV 11q13 PRDX5 4.14 × 10⁻⁰⁷ 1.33 MS 12q13 Eos (IKZF4) 3.21 × 10⁻⁰⁸ 1.34 T1D, SLE ERBB3 1.27 × 10⁻⁰⁷ 1.34 T1D, SLE 6p21.32 MICA 1.19 × 10⁻⁰⁷ 1.44 ● T1D, RA, CeD, UC, PS, SLE (HLA) NOTCH4 1.03 × 10⁻⁰⁸ 1.61 T1D, RA, MS C6orf10 1.45 × 10⁻¹⁶ 2.36 ◯● T1D, RA, PS, GV BTNL2 2.11 × 10⁻²⁶ 2.70 ◯ T1D, RA, UC, CD, SLE, MS, GV HLA-DRA 2.93 × 10⁻³¹ 2.62 ◯ T1D, RA, CeD, MS, GV HLA-DQA1 3.60 × 10⁻¹⁷ 2.15 ◯● T1D, RA, CeD, MS, SLE, PS, CD, UC, GD, GV HLA-DQA2 1.38 × 10⁻³⁵ 5.43 T1D, RA HLA-DQB2 1.73 × 10⁻¹³ 1.60 RA HLA-DOB 2.07 × 10⁻⁰⁸ 1.72 Type I diabetes (T1D), rheumatoid arthritis (RA), celiac disease (CeD), multiple sclerosis (MS), system lupus erythematosus (SLE), Graves disease (GD), psoriasis (PS), Generalized Vitiligo (GV)

TABLE 7 Common Cause Model of Skin Autoimmune Diseases. See also Jin et al., 2010, NEJM, 362: 1686-1697. Stongest association Maximum Involved in other Region Gene (pvalue) odds ratio autoimmune disease 2q33.2 CTLA4 3.55 × 10⁻¹³ 1.44 T1D, RA, CeD, MS, SLE, GD ICOS 4.33 × 10⁻⁰⁸ 1.32 T1D, RA, CeD, MS, SLE, GD 4q27 IL21/IL2 4.27 × 10⁻⁰⁸ 1.34 T1D, RA, CeD, PS 6q25.1 ULBP6 4.49 × 10⁻¹⁹ 1.65 none ULBP3 4.43 × 10⁻¹⁷ 1.52 none 9q31.1 STX17 3.60 × 10⁻⁰⁷ 1.33 none 10p15.1 IL2RA 1.74 × 10⁻¹² 1.41 T1D, MS, GD, GV (CD25) 11q13 PRDX5 4.14 × 10⁻⁰⁷ 1.33 MS 12q13 Eos 3.21 × 10⁻⁰⁸ 1.34 T1D, SLE (IKZF4) ERBB3 1.27 × 10⁻⁰⁷ 1.34 T1D, SLE 6p21.32 MICA 1.19 × 10⁻⁰⁷ 1.44 T1D, RA, CeD, UC, (HLA) PS, SLE NOTCH4 1.03 × 10⁻⁰⁸ 1.61 T1D, RA, MS C6orf10 1.45 × 10⁻¹⁶ 2.36 T1D, RA, PS, GV BTNL2 2.11 × 10⁻²⁶ 2.70 T1D, RA, UC, CD, SLE, MS, GV HLA-DRA 2.93 × 10⁻³¹ 2.62 T1D, RA, CeD, MS, GV HLA-DQA1 3.60 × 10⁻¹⁷ 2.15 T1D, RA, CeD, MS, SLE, PS, CD, UC, GD, GV HLA-DQA2 1.38 × 10⁻³⁵ 5.43 T1D, RA HLA-DQB2 1.73 × 10⁻¹³ 1.60 RA HLA-DOB 2.07 × 10⁻⁰⁸ 1.72 Type I diabetes (T1D), rheumatoid arthritis (RA), celiac disease (CeD), multiple sclerosis (MS), system lupus erythematosus (SLE), Graves disease (GD), psoriasis (PS), Generalized Vitiligo (GV).

TABLE 8 List of Genes implicated in AA. Black circles indicate genes that affect NKG2D; white circles indicate genes that affect T-cell regulation; black circles with star indicate genes that affect end organs Stongest association Maximum Region Gene Function (pvalue) odds ratio 2q33.2 ◯ CTLA4 T-cell proliferation 3.55 × 10⁻¹³ 1.44 ICOS T-cell proliferation 4.33 × 10⁻⁰⁸ 1.32 4q27 ◯ IL21/IL2 T-, B- and NK-cell proliferation 4.27 × 10⁻⁰⁸ 1.34 6q25.1 ● ULBP6 NKG2D activatign ligand 4.49 × 10⁻¹⁹ 1.65 ● ULBP3 NKG2D activating ligand 4.43 × 10⁻¹⁷ 1.52 9q31.1 ● STX17 premature hair graying 3.60 × 10⁻⁰⁷ 1.33 10p15.1 ◯ IL2RA (CD25) T-cell proliferation 1.74 × 10⁻¹² 1.41 11q13 ● PRDX5 antioxidant enzyme 4.14 × 10⁻⁰⁷ 1.33 12q13 ◯ Eos (IKZF4) T-cell proliferation 3.21 × 10⁻⁰⁸ 1.34 ERBB3 epidermal growth factor receptor 1.27 × 10⁻⁰⁷ 1.34 6p21.32 ● MICA NKG2D activating ligand 1.19 × 10⁻⁰⁷ 1.44 (HLA) NOTCH4 T-cell differentiation 1.03 × 10⁻⁰⁸ 1.61 C6orf10 1.45 × 10⁻¹⁶ 2.36 BTNL2 T-cell proliferation 2.11 × 10⁻²⁶ 2.70 HLA-DRA Antigen presentation 2.93 × 10⁻³¹ 2.62 HLA-DQA1 Antigen presentation 3.60 × 10⁻¹⁷ 2.15 HLA-DQA2 Antigen presentation 1.38 × 10⁻³⁵ 5.43 HLA-DQB2 Antigen presentation 1.73 × 10⁻¹³ 1.60 HLA-DOB Antigen presentation 2.07 × 10⁻⁰⁸ 1.72

Common pathways with other autoimmune diseases, including NK ligands in the target organs include the following:

-   -   In RA, synoviocytes aberrantly express MIC ligands, leading to         autoreactive T cell stimulation.     -   In Celiac disease, MIC ligands are overexpressed in the gut         epithelium during active disease.     -   In Type 1 diabetes, Islet cells in prediabetic NOD mice express         RAE-1 ligand.     -   The aberrant expression of NK activating ligands in genetically         predisposed individuals can induce or exacerbate disease.

Candidate pathways in AA that present new opportunities for therapy include the following:

-   -   Members of the costimulatory receptor family, which have         complementary effects on T cell activation, and their balance         controls the overall outcome of immune and autoimmune responses.     -   Genes which enhance the NK cell cytotoxic response; triggers T         cells to produce proinflammatory cytokines; and induces CD4(+) T         cells to differentiate into Th17 cells.     -   Genes which pivotal role in regulating the adaptive immune         system by controlling the survival and proliferation of         regulatory T (Treg) cells, which are required for the         maintenance of immune tolerance.     -   Activating receptors expressed on natural killer (NK) cells,         NK1.1 (+) T cells, and T cells. NKG2D ligands include MHIC class         I chain-related (MIC)A, MICB.

Candidate compounds for treatment of AA include compounds directed to Jak/Stat signaling are described in Ivanenkov et al., Mini Rev Med Chem. 2011 January; 11(1):55-78. which is hereby incorporated by reference in its entirety.

Example 2

The following genetic studies will be performed:

-   -   A replication study with another 1000 AA patients     -   Deep sequencing of candidate regions     -   Collection of 10,000 patients     -   Common Genetic Studies T1D, CeO and AA     -   An immunology study using NK activating ligands will be         performed using ULBP3, ULBP6-inducible transgenics, new mouse         models.

TABLE 9 Genes with Nominal Significance on the GWAS studies Min p- Min p- Count of value value Gene Mb SNPs <1 × 10⁻⁴ observed imputed Autoimmune Reports GO classification Chromosome 2 HDAC4 240.03 1 8.10E−05 5.59E−05 inflammatory response Chromosome 3 CACNA2D3 55.02 1 7.28E−05 1.47E−05 CeD Chromosome 5 IL13 132.02 2 1.87E−06 Asthma immune response Chromosome 6 HLA-G 29.94 1 1.07E−04 5.54E−06 RA, MS, SLE, PS, T1D, Asthma immune response HLA-A 30.01 1 1.00E−04 2.72E−05 MS, T1D, PS, GD, Asthma, Vitilago immune response MICB 31.59 2 1.89E−05 1.97E−05 MS, T1D, UC, RA, CeD, Asthma immune response TAP2 32.91 1 6.42E−06 1.28E−05 T1D, RA, SLE, PS, GD immune response Chromosome 7 IL6 22.72 2 7.72E−05 4.84E−05 RA, T1D, CeD inflammatory CHCHD3 132.44 1 3.07E−05 2.02E−05 CeD response Chromosome 8 CSMD1 3.02 1 8.65E−05 8.38E−05 CeD, MS, PS Chromosome 12 IFNG 66.84 1 1.55E−05 1.29E−05 CeD, T1D, RA, MS, SLE, PS, GD, Asthma IL26 66.87 2 7.18E−05 6.45E−05 MS, Asthma immune repsonse Chromosome 16 KIAA0350 11.11 6 1.77E−05 1.15E−05 T1D, MS, Thyroid Disease (CLEC16A) SOCS1 11.24 2 1.16E−05 8.66E−06 CeD, T1D, Asthma Chromosome 18 ANKRD12 9.25 2 3.59E−05 1.55E−05 PTPN2 12.80 1 4.09E−06 3.38E−07 CD, T1D

TABLE 10 Pathway analysis of nominal genes Category Term Genes hsa05332 Graft-versus-host disease IL6, IFNG, HLA-A, HLA-DMA, HLA-G hsa04612 Antigen processing and HSPA1L, TAP2, HLA-A, presentation HLA-DMA, HLA-G hsa04630 Jak-STAT signaling pathway SPRY2, IL6, SOCS1, IFNG, IL26, IL13 hsa05330 Allograft rejection IFNG, HLA-A, HLA-DMA, HLA-G hsa04940 Type I diabetes mellitus IFNG, HLA-A, HLA-DMA, HLA-G hsa04650 Natural killer cell MICB, IFNG, HLA-A, mediated cytotoxicity HLA-G hsa05320 Autoimmune thyroid disease HLA-A, HLA-DMA, HLA-G hsa05416 Viral myocarditis HLA-A, HLA-DMA, HLA-G hsa04060 Cytokine-cytokine receptor IL6, IFNG, IL26, IL13 interaction

Example 3—Study Samples, Genotyping, Quality Control and Population Stratification

Alopecia areata (AA) is a major medical problem and is the most prevalent autoimmune disease in the US (Table 11), affecting approximately 4.6 million people, including males and females across all ethnic groups, with a lifetime risk of 1.7% (Kyriakis K P, Paltatzidou K, Kosma E, Sofouri E, Tadros A, Rachioti E. Alopecia areata prevalence by gender and age. J Eur Acad Dermatol Venereol. 23:572-3, 2009). Additionally, AA represents the second most common form of human hair loss, second only to androgenetic alopecia, and causes significant disfigurement and psychological distress to affected individuals (FIG. 1 ). AA affects more individuals than most other autoimmune diseases combined, including lupus erythematosus (LE), type 1 diabetes (T1D), psoriasis, multiple sclerosis (MS) and rheumatoid arthritis (RA) (Table 11).

TABLE 11 Automimmune Disease Prevalence Autoimmune Disease Rate per 100,000 Alopecia Areata 1700 Psoriasis  696-1527 Rheumatoid arthritis 310-800 Type 1 diabetes 227-355 Multiple sclerosis 177-358 Systemic lupus erythematosus  34-150

In stark contrast to these other conditions, research into the pathogenesis and the development of innovative therapies in AA has lagged far behind. This can be due in part to the perception that AA is merely a cosmetic disorder. In reality, AA carries one of the highest burdens among any skin diseases, particularly among children and adolescents whose self-image is so closely linked to their appearance (Bickers D R, Lim H W, Margolis D, Weinstock M A, Goodman C, Faulkner E, Gould C, Gemmen E, Dall T; American Academy of Dermatology Association; Society for Investigative Dermatology. The burden of skin diseases: 2004 a joint project of the American Academy of Dermatology Association and the Society for Investigative Dermatology. J Am Acad Dermatol. 55:490-5, 2006).

Despite its high prevalence, there are no evidence-based treatments for AA. A comprehensive Cochrane analysis assessment of seventeen randomized clinical trials (RCTs) involving a total of 540 participants found no proven treatment of AA (Delamere F M, Sladden M M, Dobbins H M, Leonardi-Bee J. Interventions for alopecia areata. Cochrane Database Syst Rev. 2:CD004413, 2008). Each trial included from 6 to 85 participants and assessed a range of interventions that included topical and oral corticosteroids, topical cyclosporin, photodynamic therapy and topical minoxidil. Overall, none of the interventions showed significant treatment benefit in terms of hair growth when compared with placebo. It was concluded that, few, (if any), treatments for AA are proven to be effective. No RCTs on the use of diphencyprone, dinitrochlorobenzene, intralesional corticosteroids or dithranol were found, although these drugs are commonly used for the treatment of AA. Similarly, although topical corticosteroids and minoxidil are widely prescribed and appear to be safe, there is no convincing evidence that they are beneficial in the long-term. Most trials have been poorly reported and/or are so small that any important clinical benefits are inconclusive. These observations underscore the importance of defining the genetic basis of AA and determining its pathogenesis, so that rational therapeutic approaches can be developed and translational research can begin.

Pre-Clinical Evaluation of Jak/Stat Targeting in Alopecia Areata

The NKG2D receptor (NKG2D), functions to eliminate cells emitting danger signals and dysregulation of this recognition process often leads to development of autoimmunity. The influences of JAK and NF-kb inhibition will be pursued, where, without being bound by theory, NKG2DL expression will be inhibited at the transcriptional level. INCB18424, a JAK1/JAK2 inhibitor (po/topical), is in Phase II Clinical Trials for Myeloproliferative/Myelofibrosis Disorders and in Psoriasis.

TABLE 12 Small Molecule Inhibitors of Jak1/Jak2 Small molecules JAK1/JAK2 PTKi: ATP-Binding Interferon Topical Phase II/ Inhibitor Cleft Competitive Signaling Psoriasis (INCB18424) Antagonist

GWAS studies revealed a number of risk loci shared by with other forms of autoimmunity, such as rheumatoid arthritis (RA), type I diabetes (T1D), celiac disease (CeD), systemic lupus erythematosus (SLE), multiple sclerosis (MS) and psoriasis (PS), in particular, CTLA-4, IL2/IL2RA, IL21 and genes critical to Treg maintenance.

A genetic basis of AA provide avenues of exploration for therapies based on the underlying mechanisms of AA. Such therapies will focus not only on T cell subsets and mechanisms common to other forms of autoimmunity, but also on unique mechanisms that involve signaling pathways of Jak1/Jak2 and downstream effectors.

The origin of autoimmunity in AA can reside in the hair follicle itself. The studies herein are focused on defining putative danger signals in the hair follicle that contribute to the pathogenesis of AA. Pathogenic alleles that reside within the MHC, which can contribute to immune dysregulation driving the pathogenesis of AA, will also be identified. This can be due in part to the importance of antigen-presenting cells (APCs) in the presentation of danger signals as neoantigens to the immune system.

The mechanisms of Jak1/Jak2 signaling will be investigated, and the importance of Jak1/Jak2/Stat1/Stat2 interactions as drivers of disease induction will be evaluated. Pharmacologic approaches interrupting the Jak1/Jak2/Stat1/Stat2 signaling will be pursued.

C3H IFN-γ deficient mice are protected from AA development. It is shown that IFNs are capable of upregulating NKG2DL in cultured human dermal keratinocytes and fibroblasts. Further NKG2D-cross-linking induces IFN-γ production by innate NK, NKT and γδ T cells, thus creating a potential positive feedback loop, driving adaptive autoreactive immunity. Thus adoptive transfer and antibody depletion/blockade experiments will define the NKG2D-bearing cells involved.

Pharmacologic interventions aimed at interruption of the CD8 NKG2D axis will be examined. Interfering with the upregulation of NKG2DL or the activation of NKG2D-bearing cells are both rational approaches that will be pursued. It is shown that the IFN→JAK/STAT pathway can induce NKG2DL expression.

Whether the IFN→JAK/STAT pathway is pivotal to NKG2DL induction in normal and diseased skin will be assessed. The IFN JAK/STAT signaling pathway can be effectively inhibited with topical JAK1/JAK2 inhibitors, including an agent that has had early clinical success in Psoriasis. If the Type I interferon response underlies NKG2D ligand upregulation, JAK1/JAK2 inhibitors can block this induction, as well as block the IFN-gamma dependent components of the adaptive immune response.

REFERENCES

-   1. Safavi K H, Muller S A, Suman V J, Moshell A N, Melton L J, 3rd.     Incidence of alopecia areata in Olmsted County, Minn., 1975     through 1989. Mayo Clin Proc 1995; 70:628-33. -   2. Jelinek J E. Sudden whitening of the hair. Bull N Y Acad Med     1972; 48:1003-13. -   3. Ito T, Ito N, Saatoff M, et al. Maintenance of hair follicle     immune privilege is linked to prevention of NK cell attack. The     Journal of investigative dermatology 2008; 128:1196-206. -   4. Todes-Taylor N, Turner R, Wood G S, Stratte P T, Morhenn V B. T     cell subpopulations in alopecia areata. J Am Acad Dermatol 1984;     11:216-23. -   5. Gilhar A, Landau M, Assy B, et al. Transfer of alopecia areata in     the human scalp graft/Prkdc(scid) (SCID) mouse system is     characterized by a TH1 response. Clin Immunol 2003; 106:181-7. -   6. Gilhar A, Shalaginov R, Assy B, Serafimovich S, Kalish R S.     Alopecia areata is a T-lymphocyte mediated autoimmune disease:     lesional human T-lymphocytes transfer alopecia areata to human skin     grafts on SCID mice. J Investig Dermatol Symp Proc 1999; 4:207-10. -   7. Zoller M, McElwee K J, Engel P, Hoffmann R. Transient CD44     variant isoform expression and reduction in CD4(+)/CD25(+)     regulatory T cells in C3H/HeJ mice with alopecia areata. The Journal     of investigative dermatology 2002; 118:983-92. -   8. McElwee K J, Freyschmidt-Paul P, Hoffmann R, et al. Transfer of     CD8(+) cells induces localized hair loss whereas CD4(+)/CD25(−)     cells promote systemic alopecia areata and CD4(+)/CD25(+) cells     blockade disease onset in the C3H/HeJ mouse model. The Journal of     investigative dermatology 2005; 124:947-57. -   9. Ito T, Meyer K C, Ito N, Paus R. Immune privilege and the skin.     Curr Dir Autoimmun 2008; 10:27-52. -   10. McDonagh A J, Tazi-Ahnini R. Epidemiology and genetics of     alopecia areata. Clin Exp Dermatol 2002; 27:405-9. -   11. van der Steen P, Traupe H, Happle R, Boezeman J, Strater R,     Hamm H. The genetic risk for alopecia areata in first degree     relatives of severely affected patients. An estimate. Acta Derm     Venereol 1992; 72:373-5. -   12. Jackow C, Puffer N, Hordinsky M, Nelson J, Tarrand J, Duvic M.     Alopecia areata and cytomegalovirus infection in twins: genes versus     environment? J Am Acad Dermatol 1998; 38:418-25. -   13. Martinez-Mir A, Zlotogorski A, Gordon D, et al. Genomewide scan     for linkage reveals evidence of several susceptibility loci for     alopecia areata. Am J Hum Genet 2007; 80:316-28. -   14. Nakamura et al., 2008, Controlled Delivery of T-box21 Small     Interfering RNA Ameliorates Autoimmune Alopecia (Alopecia Areata) in     a C3H/HeJ Mouse Model, Am J Pathol., 172(3): 650-658. -   15. Freyschmidt-Paul, K. J. McElwee, R. Hoffmannl, J. P.     Sundberg, M. Vitacolonna, S. Kissling, M. Zoller, 2006,     Interferon-gamma-deficient mice are resistant to the development of     alopecia areata, Br J Dermatol., 155(3):515-21. -   16. Gilhar A., Kam Y., Assy B. and Kalish R., 2005, Alopecia areata     induced in C3H/HeJ mice by interferon-gamma: evidence for loss of     immune privilege, Journal Invest. Dermatol., 124(1):288-9. -   17. Wong et al., 2005, Inhibitors of the tyrosine kinase signaling     cascade for asthma, Curr. Opin. Pharmacol., 5(3):264-71. -   18. Mourich and Iverson, 2009, Splicing in the immune system:     Potential targets for therapeutic intervention by antisense-mediated     alternative splicing, Curr. Opinion Mol. Ther., 11(2): 124-132. -   19. Jin et al., 2010, Variant of TYR and Autoimmunity Susceptibility     Loci in Generalized Vitiligo, NEJM, 362:1686-1697.

Example 4

Specific autoimmune mechanisms underlying Alopecia Areata (AA) have remained obscure and therefore clinical investigation of AA has historically lagged behind other autoimmune diseases. Despite the large number of new biotherapeutics available for clinical evaluation in autoimmunity, intralesional or systemic steroids remain the standard of care and only two small randomized clinical trials have been published in the last decade. The insights provided by the discovery of GWAS genes provide a roadmap for closing this gap, by investigating GWAS-identified immunological pathways both for mechanistic and therapeutic relevance.

Identify Effective, Clinically-Relevant, Therapies in Mouse Models of Alopecia Areata. In the normal state, hair follicles (HF) represent an immuneprivileged (IP) sanctuary, expressing little or no classical HLA class I molecules. In active AA, the IP status is lost, since HLA class I expression and NKG2D ligand expression are markedly upregulated. Given the strong genetic association of NKG2D ligand loci with human AA, without being bound by theory, aberrant NKG2DL upregulation and persistent NKG2D activation on immune effector cells drives inflammatory AA initiation and progression. Without being bound by theory, in AA, as for Celiac Disease, IL-I5 can drive CD8 T cell effector differentiation enabling promiscuous “NK-type” CD8 cytotoxicity. Indeed, the local HF IL-15/NKG2DL inflammatory signals are accompanied by a dense infiltrate of CD8⁺ T cells expressing NK markers, likely the critical AA immune effectors responsible for IFNγ production and HF cytotoxicity. These collective observations, made in both the human and mouse, invite therapeutic evaluation.

The therapeutic potential of blocking NK-like reprogramming and cytotoxicity by interfering with relevant cytokines (IFN-γ□ and relevant immunoreceptor signaling pathways (NKG2D). A graft model of alopecia in which all grafted C3H mice develop AA in a timely manner, within 12 weeks, will be used. This greatly accelerates drug screening since evaluation of prevention/reversal of spontaneous disease in C3H/HeJ is otherwise impractical given the late onset (>6 months of age) and low cumulative incidence (15%). Both small molecule approaches, that have the advantage of potential topical delivery, and biotherapeutic proteins (antibodies) that have superior biologic specificity, will be evaluated. The overarching goals are to use biotherapeutics to establish critical immune pathways that might be targeted by more clinically tenable, small molecule topical approaches.

Identify disease-associated AA biomarkers and their reversal with effective therapy in C3H mice. Approaches using GWAS and transcriptional profiling, for example, as well as approaches using immunostains and FACs of T cell subsets, for example, have been taken to identify several pathogenic AA biomarkers in the skin and blood of AA mice. These mouse studies will continue to inform developing a translational biomarker platform in human AA. In the skin, the studies in alopecic C3H/Hej mice demonstrated that IFN-induced genes are dramatically upregulated, including the chemokines CXCL9-11, which are likely responsible for recruitment of a dense infiltrate of HF-associated IFN-γ-producing CXCR3⁺ NKG2D-bearing CD8 T effectors. Spectratype data provide striking evidence that these circulating NKG2D⁺ CD8 T effectors are bonafide autoantigen-driven effector T cells that increase with disease progression. Temporal assessment of these serologic, cutaneous and cellular biomarkers in mice during treatment will enable one to identify dynamic, mechanistically important, prescient inflammatory biomarkers that predict therapeutic outcome and inform clinical biomarker discovery/utilization.

Preclinical validation of targeted approaches using AA human tissues ex vivo. To expedite clinical development of treatments that demonstrate “proof-of-concept” in the AA mouse model, human AA tissues will be used for target validation and correlative studies. The “crawl-outs” system will provide a convenient ex vivo human bioassay to validate that agents that reverse mouse AA translate to the human, effectively inhibiting human AA T cell survival/expansion/function.

Just as the human DNA from thousands of alopecic subjects provided GWAS genes and unique insight into the human disease, human tissues, blood and skin from AA subjects will be used to continue to interrogate these genetically implicated immune pathways ultimately for therapeutic utility. Conceptually, the mouse model of alopecia areata this human pursuit, providing the pre-clinical testing grounds for therapeutic approaches in alopecia areata.

Autoimmune diseases affect an estimated 23 million individuals in the US (1) and although the prevalence of AA is not established, it is one of the most prevalent autoimmune diseases (2, 3). In 1999-2000, there were an estimated 2.4 million office visits for AA, half of which by patients in their 20s and 30s. AA causes significant disfigurement and psychological distress to affected individuals and carries one of the highest burdens among any skin diseases, particularly among children and adolescents whose self-image is so closely linked to their appearance (4). At present, the prognosis of AA is unpredictable and there is no definitive treatment. Current therapies include steroids and topical immune therapy, and induce durable remissions in only one third of patients (5, 6). Despite its high prevalence, a comprehensive Cochrane analysis assessment concluded that there are no evidence-based treatments for AA (7).

In stark contrast to other autoimmune conditions, research into the pathogenesis and the development of innovative therapies in AA has lagged far behind. In the era of targeted immunological therapies, only two clinical studies evaluating protein biotherapeutics in AA have been reported (8, 9) (both targeting T cell trafficking). In part this has been due to the limited understanding of specific AA immune pathways that would prompt evaluation of rational therapeutic approaches. Based on new understanding of the genetic and pathogenic basis of AA pre-clinical proof-of-concept data that will prompt clinical evaluation of rational therapeutic approaches in this disease will be obtained.

The GWAS (10) study revealed a number of risk loci shared by with other forms of autoimmunity, such as RA, T1D, celiac disease (CeD), SLE, MS and PS, in particular, CTLA4, IL2/IL2RA, IL21, NKG2D Ligands and genes critical to Treg function (Eos). The genetic commonality with RA, T1D, and CeD is especially noteworthy in light of the pathogenic significance of the expression of an NK ligand in the end organ (synovium, islet, gut and skin), and the involvement of the NKG2DL/NKG2D pathway in the pathogenesis of each of these three diseases (11, 14). One advantage of immunotherapeutic studies in the skin is the relative ease of access of the target organ. Thus, the studies herein examining the skin can provide important insight into NKG2D and rn-15 triggered CD8 cytotoxicity, IFN-triggered injury, etc., that impact the understanding of these other related human diseases in which the target organ is not accessible. Indeed positive studies in any one of these autoimmune diseases that share a common cause can serve as the basis for common treatments.

Specific autoimmune mechanisms underlying AA have remained obscure beyond the prevailing view that it is the result of T cell mediated attack of the hair follicle. In the normal state, hair follicles represent an immuneprivileged (IP) sanctuary (15-17), expressing little or no classical HLA class I molecules and instead express the inhibitory HLA ligands HLA-E and HLA-G, thus simultaneously avoiding immune recognition by class I restricted CD8 cells while negatively regulating NK cells, that might otherwise react to “missing self” (16, 18-21). In active AA, the IP status of the normal hair follicle is not operative, since HLA class I expression is markedly upregulated. Previous studies have noted both CD4 and CD8 T cell infiltrates surround the hair follicle in alopecic areas of skin in the mouse and human (22, 23). Indeed transfer of total T cells, but not B cells or sera, can transfer disease from an alopecic mouse or human to a normal WT (24) or SCID mouse (25), implicating effector T cells as the pathogenic cells. However molecular mediators of site-specific inflammation have not been previously identified. Guided by the GWAS findings and previous immunobiological studies, new insight into this common autoimmune disorder is provided, that will lead to the development of innovative, rationally targeted treatments to fill the unmet needs of patients with AA.

AA Immunological Pathways Identified by GWAS Studies The most highly-significant genetic loci found associated with alopecia areata are shown in Table 13. These seminal studies have been validated in a confirmatory study in a distinct population. Underlined genes are HF expressed genes while bold genes are genes expressed in immune cells. Overall, many susceptible loci are shared by other tissue-specific autoimmune states, with Type I diabetes exhibiting the most striking overlap (right column). The commonality with RA, T1D and CeD is particularly noteworthy, as NKG2D has been shown to have a significant role in the pathogenesis of each of these three diseases (11-14). Thus insight in any one of these disease/model systems is likely to shed light on common pathogenic pathways shared by multiple autoimmune states. The most highly significant alleles identified (other than HLA) were four NKG2D ligands (UBLP-3/UBLP-6/MICA/MICB in Bold italics) and one inhibitory ligand (HLA-G). The NKG2D receptor (NKG2D), functions to eliminate cells emitting danger signals and dysregulation of this recognition process often leads to development of autoimmunity (26). Other HF expressed genes that contribute to risk include HLA, antigen processing genes (TAP) and PRDX/STX17.

TABLE 13 Genetic loci associated with AA. 2q33.2 CTLA4 Inhibits 3.6 × 10⁻¹³ T1D, RA, CeD, costim MS, SLE, GD ICOS T-cell costim 4.3 × 10⁻⁰⁸ T1D, RA, CeD, MS, SLE 4q27 IL21 NK function/ 4.3 × 10⁻⁰⁸ T1D, RA, CeD, Th17 PS IL2 T-cell./Treg 3.0 × 10⁻⁰⁴ T1D, RA, CD, prolif MS, CD, SLE, PS, GD 6q25.1 ULBP6 NKG2D ligand 4.5 × 10⁻¹⁹ None ULBP3 NKG2D ligand 4.4 × 10⁻¹⁷ None 9q31.1 STX17 hair graying 3.6 × 10⁻⁰⁷ None 10p15 IL2RA T-cell 1.7 × 10⁻¹² T1D, MS, GD prolif./Treg 11q13 PRDX5 antiox enz 4.1 × 10⁻⁰⁷ MS 12q13 Eos T-reg 3.2 × 10⁻⁰⁸ T1D, SLE programming 6p21.3 MICA NKG2D 1.2 × 10⁻⁰⁷ T1D, RA, CeD, (HLA) ligand UC, PS, SLE NOTCH4 Hematopoiesis 1.0 × 10⁻⁰⁸ T1D, RA, MS BTNL2 T cell 2.1 × 10⁻²⁶ T1D, RA, UC, costim CD, SLE, MS HLA-DRA Ag 2.9 × 10⁻³¹ T1D, RA, CeD, presentation MS HLA-DQA1 Ag 3.6 × 10⁻¹⁷ T1D, RA, CeD, presentation MS, SLE, PS, CD UC, GD HLA-DQA2 Ag 1.4 × 10⁻³⁵ T1D, RA presentation HLA-DQB2 Ag 1.7 × 10⁻¹³ RA presentation HLA-DOB Ag 2.1 × 10⁻⁰⁸ SLE presentation  5 IL-13 Th2 1.9 × 10⁻⁰⁶ Asthma polarization  6 HLA-G NKG2A 1.0 × 10⁻⁰⁴ RA, MS, SLE, inhibition PS, T1D, Asthma HLA-A Ag 1.0 × 10⁻⁰⁴ MS, T1D, PS, presentation GD, Asthma, Vitiligo MICB NKG2D 1.9 × 10⁻⁰⁵ MS, T1D, UC, ligand RA, CeD, Asthma TAP2 Ag 6.4 × 10⁻⁰⁶ T1D, RA, SLE, presentation PS, GD  7 IL6 Inflammation 7.7 × 10⁰⁵  RA, T1D, CeD 12 IFNG Th1/inflamm 1.5 × 10⁰⁵  CD, T1D, RA, MS, SLE, PS, GD, Asthma IL26 T 7.2 × 10⁻⁰⁵ MS, Asthma polarization 16 CLEC16A C-type lectin 1.8 × 10⁻⁰⁵ T1D, MS, GD receptor SOCS1 JAK/STAT inh 1.2 × 10⁻⁰⁵ CeD, T1D, Asthma 18 PTPN2 phosphatase 4.2 × 10⁻⁰⁶ CeD, T1D

IFN-γ production by activated NKG2D-bearing CD8 effectors can reverse immunologic privilege and perpetuate the inflammatory loop.

IFN-γ related pathways can be undermined with existing therapeutics under clinical investigation, expediting the path to translation. Over the last ten years, several small molecule inhibitors of protein tyrosine kinases (PTKi) have been successfully developed clinically both for oral and topical delivery. PTKi approaches will be pursued to blocking CD8 mediated inflammatory responses in AA, namely inhibition of JAK/STAT signaling, responsible for cytokine responsiveness, downstream of the CD8 effector cytokine IFN-γ.

Identify Clinically-Relevant Therapies Targeting Effector T Cell Responses in Alopecic Mice.

Using immunostaining and flow cytometric analysis of total cellular populations from AA skin, CD8⁺ NKG2D⁺ T cells were found infiltrating the hair follicle (FIG. 17 ) and expanded in AA but not normal skin (FIG. 18 and FIG. 4A). Furthermore AA mice exhibit cutaneous lymphadenopathy, at least in part due to dramatic systemic expansion of the CD8⁺ NKG2D⁺ population, in both the AA cutaneous lymph node and in the blood, representing fully 6+/−1% of total LN cells are 2.4+/−1.3% of total PBMCs, each more than ten-fold higher than that seen in normal C3H mice. These CD8⁺ NKG2D⁺ T cells are potently cytotoxic against HF dermal sheath cells (FIG. 21 ). Further immunophenotypic analysis of CD8⁺ NKG2D⁺ T cells revealed that this population expressed the T memory marker CD44⁺ and multiple “NK markers”, including DX5, NKG2A/C/E, and NKp46 (FIG. 19 ).

To provide proof that the circulating CD8⁺ NKG2D⁺ population represented the bona fide “AA-specific” effector population, without being bound by theory, the CD8⁺ NKG2D⁺ T cells found in the lymph node can express a similar TCR repertoire to the total T cells found infiltrating AA skin. FIG. 20 shows that CD8⁺ NKG2D⁻ lymph node populations exhibit a normal distribution of CDR lengths, a pattern reflective of an unrestricted polyclonal T cell population. In contrast, the TCR repertoire of skin T cells are highly restricted with nearly all Vp family members dominated by a small number of CDR lengths, indicative of an oligoclonal population of antigen-driven T cells. CD8⁺ NKG2D⁺ T cells from the cutaneous lymph node cells are also highly restricted and show a strikingly similar pattern with total cutaneous T cells.

These data confirm that CD8⁺ NKG2D⁺ T cells contain most of the oligoclonal T cell populations found in the skin. Moreover the evidence for the expansion of common oligoclonal T cells in the skin and lymph node argues against the possibility that these LN populations (or even markers) are expanded/activated as a reactive process to inflammatory events. For instance, Treg cells (CD4⁺ CD25⁺ FoxP3⁺) are also increased in numbers in AA lymph nodes, however, this is likely a reactive process, since their spectratype is diverse and unlike those present in the skin. Note that some TCR/clones are found in skin but not in draining cutaneous lymph nodes, e.g., Vβ10. These likely include CD4⁺ T cell populations found in AA skin but not present in the sorted CD8+LN fraction. These data establish that the CD8⁺ NKG2D⁺ T cells are the pathogenic NKG2D⁺ effectors in mouse AA, establishing a parallel of the mouse model to human AA.

Pre-Clinical AA Model to Establish Proof of Concept for Therapeutic Approaches In Vivo

C3H/HeJ Skin Graft Model: The C3H/HeJ mouse model for alopecia areata has been studied since it was first reported this model in 1994 (29, 30, 31). While other mouse models for alopecia areata were found (32), this model remains the most studied and used in the field today. Having done a variety of drug trials with this model over the years (33), a variety of protocols have been optimized (34).

Full thickness skin grafting from alopecic C3H/HeJ mice to 2-3 month-old unaffected C3H/HeJ female mice results in patchy alopecia within 10 weeks of grafting in all recipients, progressing to generalized alopecia at 20 weeks. A variety of comprehensive and efficient histological scoring systems are employed building on the traditional training of histopathologists where the adjective used in descriptions (normal, 0; mild, 1; moderate, 2; severe, 3; and extreme, 4) are converted to a numerical value for importation into a relational database (Mouse Disease Information System [or MoDIS]: research.jax.org/faculty/sundberg/registration.php) (35, 36). This database is exportable via Excel for importation into a statistical analysis or other software program. Alopecia areata is graded based on severity, location of inflammatory cells, types and relative numbers of inflammatory cells (granulocytes, mast cells, lymphocytes), follicular dystrophy, etc. Molecular RNA and protein biomarkers will be incorporated in this panel, and generation and analysis of this multiple variable database will be done.

Protocol #1: In the prevention setting, recipient mice will be treated beginning the first week after grafting.

Protocol #2: For treatment, grafted mice with early-established AA will be treated to reverse AA progression.

Agents will first be examined in the prevention model. Agents that successfully prevent AA will then move forward for secondary evaluation in the setting of established disease, while those that fail the first test will be discarded, permitting entry of other innovative approaches. The following are the initial therapeutic approaches, both specific biologics and small molecules, chosen for preclinical evaluation.

Small molecule JAK protein tryosine kinase inhibitors (PTKis) that target activation pathways involved in the T effector response will be tested. These PTKis are already in Phase III clinical trials easing translation and potential clinical development of “NK-type” T cell-targeted topicals.

Therapeutic Interventions with Small Molecule PTKi Interventions

Topical delivery has obvious advantages in limiting systemic exposures and toxicities. Pre-clinical and clinical studies have demonstrated that small molecule PTKi delivery in cream can overcome the skin barrier and achieve effective local concentrations in the dermis with limited systemic exposure (78). Key PTKs pivotal to effector T responses in AA will be targeted, namely the cytokine IFNγ.

Targeting IFNs with Jak1/2 Inhibitors: Interferons are an attractive therapeutic target in AA since they likely participate at several steps in the inflammatory response; including eliminating HF immunologic privilege and inducing the cellular inflammatory response. Interferons upegulate several relevant pro-inflammatory molecules in the hair follicle end organ, including NKG2DL, adhesion molecules (e.g., ICAM-1), antigenic processing/presentation (TAP1/2, LMP, proteosome, MHCI and II) and moreover drive the immune effector response (increased Th1-type responses, DC activation and IFN-γ-mediated cytotoxicity). In the C3H-HeJ mouse model of AA, IFN-γ is required for pathogenesis (89, 90) and administration of interferon-y accelerates disease (91). Importantly, administration of IFN-γ neutralizing antibodies reverses AA pathogenesis in the C3H-HeJ mouse (92, 93). Likewise, in the human, alopecia areata has been noted in several series as a side-effect of Type I interferon therapy (94-104) and transcriptional profiles of human AA skin have noted a Type I IFN response in lesional biopsies (105) and Th1 skewing and elevated IFN-response cytokines/chemokines in the peripheral blood (FIGS. 22, 23 ) (106-110) and reviewed in (111). T cells obtained from AA skin biopsy explants were uniformly CD8⁺ NKG2D+ cells and when stimulated produced IFN-γ (FIG. 21A) but not IL-4 or IL-17. In stark contrast, the majority of T cells obtained from normal skin explants are CD4⁺ (112, 113). The situation is similar in AA mice, IFN-γ producing CD8 T cells are expanded in the cutaneous lymph node (FIGS. 19, 21 ). In the skin, our transcriptional profiling studies of total skin isolated from lesional vs. non-lesional C3H skin has identified an interferon response as the dominant signature with 17 of the top 20 upregulated genes being interferon response genes (FIG. 22 ). Indeed, IFNs upregulate NKG2DL expression on hair follicle targets, closing an IFN-mediated pathogenic circle, in which IFN-γ made by CD8 effectors drive the HF NKG2D ligands that lead to CD8⁺ NKG2D⁺ activation.

Clinical JAK1/2 inhibitors: Both blocking antibodies (114, 115) and small molecule PTKis are under clinical development targeting the IFN response in autoimmunity. IFN induced STAT1/2 activation is mediated by Jak1/Jak2/Tyk kinases and clinical data have emerged using oral Jak1/Jak2 PTKis in several autoimmune conditions, including RA and psoriasis. Topical therapies have the added advantage of an improved therapeutic index limiting the potential for systemic immunosuppression. INCB018424 inhibits Jak1 and Jak2 (IC₅₀=1 nM) and is the only topical JAK inhibitor (78) currently in clinical investigation and its safety profile to date has been acceptable. Proof-of-concept with oral delivery has been demonstrated in a Jak-dependent disease (myelofibrosis) (116) and in a dermatological disease driven by local cytokine production (psoriasis models) (78). In psoriasis subjects, topical INCB018424 induced rapid clinical responses and normalization of cutaneous Th1/Th17 cytokine responses (unpublished data from clinicaltrials.gov/ct2/show/NCT00820950). Taken together, topical INCB018424 is a safe intervention predicted to have efficacy in Alopecia Areata, an inflammatory state dominated by an IFN-signature.

Approach: Protocol #1: Systemic delivery of INCB018424: Twice daily (90 mg/kg) orogastric gavage provides good systemic exposure given the t_(1/2) half-life of 3-6 hours in the rodent. In this manner we can assess if JAK1/2 inhibition prevents AA progression.

Protocol #2: Topical delivery of INCB018424 (1.5%): Reversal of patchy AA by topical JAK1/JAK2 inhibitor. Daily topical treatment (78) will begin to new affected patchy areas in grafted mice that begin to emerge 6-10 weeks after grafting. Topical treatment can be restricted to specific areas of mouse skin by appropriate bandaging to prevent systemic exposures from grooming, licking etc. Importantly, by treating affected areas we can assess the clinically relevant question of whether topical exposure can reverse AA progression.

Identification of Murine Biomarkers of AA Disease Activity that Inform Clinical Investigation

The identification of immunological events that accompany treatment outcome in these pre-clinical studies, is important, both to validate the proposed mechanism of action and to provide useful biomarkers that will inform potential clinical evaluation of these same therapies in AA patients. Since in clinical evaluation, biospecimens will be limited to blood and skin samples, the focus will be on mouse biomarkers in the mouse that are easily sampled from these sites. Approaches using GWAS and transcriptional profiling, for example, as well as approaches using immunostains and FACs of T cell subsets, for example, have been taken to identify several pathogenic biomarkers in the skin and blood of AA mice. The studies in alopecic C3H/HeJ mice have already identified several skin biomarkers upregulated in AA, such as IFN-induced genes (FIG. 22 ). Spectratype data (FIG. 20 ) has provided striking evidence that these circulating NKG2D⁺ CD8 T effectors are bonafide autoantigen-driven effector T cells. Temporal assessment of these serologic, cutaneous and cellular biomarkers in mice, with and without treatment, will enable one to identify dynamic, mechanistically important, prescient inflammatory biomarkers that predict therapeutic outcome and inform clinical biomarker discovery/utilization.

Cellular biomarkers of circulating immune effectors in AA: “NK-type” CD8 T cells are expanded in the skin, blood and lymph node in AA mice and are a valuable tool to investigate pathogenesis. Moreover, the cellular subset appears directly relevant to the human disease.

Spectratype-based imunomonitoring: NK-type CD8 T cells will be identified in AA mice in the circulation by flow cytometry of 50 μl of whole blood (FIG. 18 ). The total numbers and spectratype of this CD8⁺ NKG2D⁺ sorted T cell subset, will be assessed every three weeks after grafting to monitor the evolution of clonal dominance/epitope spreading within a single mouse. Spectratype analysis will be used to confirm that immunophenotypic loss of CD8⁺ NKG2D⁺ T cells is due to treatment-related elimination of the circulating alopecic T cell clones. This non-invasive immunomonitoring approach will be used to follow mice on all treatments. For instance, 3 weeks of treatment with the JAK1/2 inhibitor INCB018424 did not. Without being bound by theory, JAK1/2 inhibitors are expected to abrogate the downstream inflammatory consequences of T cell derived effector cytokines (IFN-γ, IL-17) in the target tissue.

Immune “proteomic” signatures in serum: Cytokines and chemokines in the serum are a reflection of the inflammatory milieu in the target organ. The data in the human demonstrate elevations of several cytokines and chemokine some of which correlate with disease activity (for instance IL-8), extending the observations of prior studies (106, 107, 109). Consistent with our overall findings implicating IL-15 and IFN-γ in AA pathogenesis, it is seen that IFN-γ and the well-known IFN-γ inducible chemokine IP-10 (CXCL10) are upregulated in human AA sera. Functionally relevant inflammatory cytokine/chemokine biomarkers will be identified in the sera from AA mice using a multianalyte approach (LUMINEX™ platform). The advantage of biomarker development in the mouse, besides genetic homogeneity, is the relative ease of longitudinal studies to identify biomarkers that occur early or late in disease progression and conversely with treatment response. At the RNA level, C3H mouse skin IFN microarrays demonstrated that both Type I IFNs (IFNα₁₋₃, ε□ and type II IFN (IFN-γ□ were upregulated as well several IFN-associated chemokines (CXCL9-11, CCL5). Cytokine/chemokines production will be examined in sera using custom multianalyte LUMINEX™ assay (R&D), comprehensively addressing serum levels of 9 chemokines (CCL2-5, CCL11, CXCL5, CXCL9, CXCL10, CXCL11) and 14 cytokines (IFNα, IFNγ, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-15, IL-17, TNF, GM-CSF). We will include antibody pairs to detect serum levels of NKG2DLs (117) and IL-15Ra.

Transcriptional profiling of skin: In human AA, published studies of small series of patients have noted a Type I IFN response in lesional biopsies (105) and Th1 skewing and elevated IFN-response cytokines/chemokines in the peripheral blood (106-110) and reviewed in (111). These data are mirrored in the analysis of the C3H mouse. Transcriptional profiling of RNA isolated from alopecic mouse skin revealed a dominant IFN response. 18 of the top 21 upregulated genes in AA skin were IFN response genes, which was confirmed by real-time PCR (FIG. 22 ). The upregulation of the IFN-response genes CXCL9-11 are notable since the CD8⁺ NKG2D+ cells also uniformly express their shared chemokine receptor, CXCR3, a receptor upregulated on short-lived immune effectors (118-120). These chemokines are also upregulated in the sera of human AA subjects and thus can be a functionally relevant AA skin biomarker in the skin (mRNA) and in the blood (protein). Work in this area will continue, building a database of skin RNA signatures collected before and during ongoing treatment, that can be used to tailor treatment plans and as early indicators of treatment response. Towards these goals, parallel studies will be performed in the mouse with both intralesional steroids and CTLA4-Ig, as well as with therapeutic interventions, to provide a transcriptional profile database that will be useful in cross-referencing the human treatment studies (comparative genomic analysis between studies and for potential clinical development of more novel approaches).

Human Subjects

Skin biopsies from the scalp will be collected from a minimum of 100 Alopecia Areata patients and approximately 100 controls. The subject population will consist of AA patients and unaffected control subjects receiving hair transplantation. Approval for collection of these samples underwent expedited review/exemption since they are discarded tissue. A protocol for collection of scalp biopsies from AA patients is in preparation. A minimum of 100 AA subjects will be recruited, but collection of greater than 100 will be beneficial and increase the power of this approach.

In order to achieve maximum homogeneity among samples for generating the expression interactome, hair follicles will be collected exclusively from middle aged Caucasian male subjects with AA, since the matching control donors are most commonly from this group. Therefore, despite the fact that AA shows no ethnic or gender predilection, the AA patients will be selected to match in age and gender to the controls for the purpose of this study (middle aged Caucasian males). Patients with active disease (ie: with some remaining hair follicles) will also be selected so that they can be efficiently microdissected.

Vertebrate Animals

Young C3H/HeJ mice (2-3 months) and retired breeders showing visible hair loss will be acquired from Jackson Labs. The animals will be kept under standard conditions in the animal facilities.

The mice will be administered drugs or antibodies by either systemic injections or topically. For topical applications, the mice skin will be shaved on the dorsal surface with electric clippers. One week after shaving, mice will receive topical application of the chemicals dissolved in acetone or 10% v/v in propylene glycol as vehicles. Injections will be given either intraperitonealy or subcutaneously. The dosage as well as frequency of administration will be followed as per established protocols. The mice will be monitored daily for signs of distress throughout the duration of treatment.

REFERENCES

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Example 5

Alopecic effector T cells were identified in the lymph node and blood of alopecic mice using spectratyping of flow sorted cells. A blood-based assay was developed to monitor these alopecic T cells during treatment. Cytotoxic assays have been developed for evaluating functional components of the hair follicle (HF) interactions with CTLs, in both human and mice.

Using human biospecimens obtained from AA subjects, it was demonstrated that circulating T cells express high levels of NKG2D and primary T cells obtained from the skin are dominated by a CD8⁺ NKG2D⁺ IFN-γ producing cells establishing parallels between the mouse and human disease.

Immunophenotyping and Skin Immunobiology

To study pathways in biospecimens requires analysis of heterogeneous cell populations in the skin and in the blood. In the clinical investigation of human autoimmunity, alopecia areata provides a unique opportunity because of the accessibility of the end-organ enabling study/isolation of the pathogenic immune effectors in their relevant microenvironment. Analytic tools and personnel required will be provided for optimal study of these precious end-organ skin biospecimens, and their cellular/serological counterparts in the blood obtained from the same individual.

Preparation and Analysis of Biospecimens:

The following will be provided to assist in the analysis of blood:

i) Multianalyte cytokine/chemokine analysis from serum samples. ii) Flow cytometric immunophenotyping, including cytokine analysis of human PBMCs from AA subjects. iii) Flow sorting for downstream applications using T cell subsets (RNA profiling, spectratyping).

The following will be provided to assist in the analysis of skin:

-   -   i) Preparation/analysis of viable cellular fractions of both         primary hair follicle and T cell components for functional         (cytotoxicity) and analytic studies (T cell cloning,         spectratyping).     -   ii) Immunostaining and transcriptional profiling of whole skin     -   iii) Flow cytometric immunophenotyping of dermal lymphocytes         from human and mouse AA skin.

Scientific Experiments

Pathogenic HF-specific cellular subsets in the peripheral blood of AA subjects will be identified. To this end, spectratype analysis will be used as a tool to identify circulating human alopecic T cells populations, as was done for the mouse model, by matching the spectratype found in total skin T cells with the spectratypes of specific sorted peripheral blood T cell subsets in the peripheral blood obtained from the same patients.

Th1-predominance in human AA will be established and the rationale and biomarker platform for Th1-targeted therapies will be developed. Therapeutic targeting of disease-specific Th-pathways has succeeded in other human skin inflammatory diseases, most notably in psoriasis a prototypical Th17 disease. There is substantial data in the AA animal model for AA as a predominant Th1 disease. The Th-profile of infiltrating dermal AA T cells and relevant circulating T cell subsets in the blood will be addressed using a multi-prong approach, including multianalyte, RNA profiling, immunstaining and intracellular flow cytometric analysis from the skin and blood.

An integrative approach to translational research begins with basic studies identifying targetable pathways in AA, then testing these pathways therapeutically in preclinical models, longitudinal biomarker assessment in clinical trials and pursuing population-based research based on genetic studies. The pre-clinical therapeutic effects of interventions will be evaluated in the grafted AA mouse models, treatment effects on the inflammatory responses will be assessed in the skin and blood and human biospecimens will be provided for validation of human relevance in ex vivo studies.

Monitoring the pathogenic interactions between immune cells and target cells in the skin in humans requires sophisticated processing and analysis of primary human tissues and blood.

Flow cytometry has become the primary tool for the identification of cell populations according to specific parameters, and is therefore employed by an ever-growing number of biomedical scientists.

The immunobiology of the skin presents specific characteristics and challenges; the special architecture of the tissue including its barrier functions, its associated unique immune cell populations and its close proximity/interactions with microbial flora, and the practical difficulties of isolating infiltrating lymphocytes, are all part of the complexities needed to be understood in the approach to immunological diseases of the skin. Services that provide assistance with tissue processing and staining, as well as cell isolation, and essential to insuring high quality consistent results are available.

Flow cytometry is a tool for clinical investigators and a working knowledge of its use is required for all translational immunologists. Four flow cytometers, including two workhorse instruments (FACs CANTO™ and FACs CALIBER™) a 6-laser LSR II™ and a 4-laser BD INFLUX™ are available. The LSR II™ and BD INFLUX™ have been designed to have comparable lasers/detectors to facilitate transition from analysis to preparative sorting. The LSR II™ is equipped with 6 lasers, capable of detecting in total 19 colors simultaneously providing versatility to detect both fluorescent proteins (mBanana, GFP, BFP, RFP/dsRed, mCherry mRasberry) and fluorescent dyes/chemical fluorochromes. LSR II™ can analyze diverse cell types within heterogeneous cell populations from tissues using a wide array of organic and inorganic fluorochromes. These additional fluorescent parameters allow detection of coordinated functional events in specific cell types in mixed populations (e.g. intracellular IFN-gamma, IL-10 production and phosphoprotein detection in T cell populations from cutaneous tissues and draining lymph nodes). The basic LSR II™ instrument is equipped with three lasers; blue (488 nm), red (633 nm), violet (405 nm). The custom designed LSR II™ contains in addition, three additional lasers permitting up to 20-color detection and a 96-well plate reader. “Leave one out” 10-color panel designs for T cell subsets (for example, Tregs), B cells and monocyte have been designed to take advantage of the 100W yellow-green 594 laser which has exquisite sensitivity for “red” ALEXA™ 594. Thus, investigators may adopt a “leave-one out” design to sensitively examine expression for a specific marker of interest by staining with ALEXA™ 594 conjugated antibodies.

Quality: Panel development for immunophenotyping lymphocyte populations in human peripheral blood and other experiments will be conducted. Because of the capacity of the 6 laser system of the LSR II™, a common “easy-to use” set of colors that can be used without the need for compensation was established. The ability to do a 5/6-color experiment without the need for fluorescent compensation gives increased data resolution as the fluorochromes as far apart from each other in the spectrum, and excited independently by each laser line. This allows for the ability of populations to clearly separate, something that is rare when compensation is applied. The current selected fluorochrome combination is DAPI (gated on the DAPI negative cells) Pacific Blue, FITC, PE, ALEXA FLUOR™ 594 and APC, however other combinations of fluors are currently being tested and used. This 5-flourochrome set serves as the base platform for panels, to which additional fluorochromes can be added. In a proof-of-concept experiment, the use of 5 flurochromes was shown simultaneously without the need for compensation. C57BL/6 splenocytes were stained with CD8 Pacific Blue, CD3 FITC, CD45R (B220) PE, CD11b Bio+ Streptavidin ALEXA FLUOR™ 594, and CD4 APC.

Maintenance and Quality control for Skin Immunobiology: The procedure used for the isolation of lymphocytes from skin biopsies has been adapted from a method developed by Clark and Kupper (2) in which biopsies are culture on 3-dimensional tantalum-coated cellfoam matrices to promote migration of T cells from the biopsies. Of each isolated cell populations a minor fraction will be used for CD45/CD3 staining to assess the number of leucocytes/T lymphocytes by flow cytometry prior to further studies or cryopreservation. For re-use, the matrices will be soaked for 30 min in 10% bleach, then rinsed in water and transferred into a solution of ENZYTE™ enzyme cleaner (Decon Labs). The matrices will be left in this solution on a hot plate with stirrer for 24 hrs, rinsed with distilled water and let dry before autoclaving.

Whole skin analytic techniques (e.g., immunostaining) will be provided. The following will be provided: 1) critical expertise in skin immunobiology for the preparation/analysis of viable cellular fractions of both primary hair follicles and cutaneous T cells; 2) biomarker development capabilities for clinical biospecimens including multianalyte analysis (LUMINEX™) and transcriptional profiling of precious clinical biospecimens (skin and blood from subjects enrolled in clinical trials). The tools to investigate biological processes and monitor the inflammatory AA mechanisms in pre-clinical and clinical samples from both the blood and the AA target end organ, the skin will be provided.

Capabilities in microarray data analysis and storage, sequence and pathway analysis are available for the studies herein, and extend all the way to the most recent algorithms for regulatory network reverse engineering. A number of widely-used databases are created and maintained. In addition, all important sequence and structure databases are maintained centrally. This allows direct large-scale searches, if need be using custom algorithms and cluster computing. Many of the research methods developed are packaged into software applications and computational services, freely available to the scientific community (c2b2.columbia.edu/page.php?pageid=10).

The following services are available:

1. Immunomonitoring:

a. Multianalyte Cytokine & Chemokine Analysis from Serum Samples

Two approaches are available: a) The multiplex bead based immunoassays systems (e.g. CBA/FLOWCYTOMIX™) provides multiple analytes that allow 10 or more cytokines/chemokines to be assessed on a single read of a small volume (50 μl) sample. The LSR II™ 96 well high throughput capacity provides ease of use combined with efficiency. Software (“snap-to” gating) is available in house for data analysis. A multiplex bead-based assay system will also be developed to quantify soluble human and murine NKG2DL in the sera, for which specific antibody sets are commercially available. b) Multiplex analysis using the LUMINEX™ platform is available in the CTSA for analysis of commercially available mouse and human cytokine and chemokine arrays (FIG. 24 ). The LUMINEX™ assays are provided on a fee-for-service basis, with reagents purchased by the user. Once an AA serum biomarker platform bulk purchase of selected analytes will enable cost-savings.

Flow Cytometric Immunophenotyping

It has been established using multiparameter flow that CD8+ NKG2D+ cells dominate the dermal leukocyte population harvested from the skin of mice with spontaneous alopecia (FIG. 25 ). To address the human situation, flow panels have been developed to define quantitative alterations in any of 30 PBMC subsets focusing on NK and CD4 and CD8 T cell populations using 3 FACS tubes (T cell subset tube, NK-marker tube for CD4, CD8 and NK cells and a third non-T cell tube); Lineage Markers: CD3, CD4, CD8a, CD80, CD14, CD19, CD20, CD56, CD64; T cell subset Markers: CD45Ra, CD45Ro, CD56, CD62L, CCR7, CD127, FoxP3, Helio; Cutaneous Chemokine Receptor Analysis: CCR4, CCR8, CCR10, CLA, E and P-selectin; NK immunoreceptor family members: Inhibitory: KIR/CD158, CD94/NKG2A, LILR/ILT, LAIR1 NKR-P1 Activatory: NKp46, NKp30, NKp44, NKG2C, NKG2D. In the initial evaluation of PBMCs from four AA subjects, increased NKG2D expression was observed on CD56+CD8+ T cells and NK cells, but not on CD4+ T cells (FIG. 26 ).

Cytokine analysis: Activation of freshly isolated buffy coat peripheral T cells with PMA/ionomycin will drive cytokine production by circulating memory T cells. After a 4 hour incubation with brefeldin, cells are cell surface marker stained, fixed and permeabilized prior to intracellular staining for IL-2, IFN-γ, TNF, IL-4, IL-17, and FOXP3/helios. These cytokines/transcription factors together with the surface markers CD3, CD4, CD8, CD25, CD56, CD62L, NKG2D, CD45Ra, CD45Ro, CLA/CCR4 will delineate the fraction of Th1/Th2/Th17/Tregs in the total and cutaneous CD4/CD8 naïve and memory T cell compartment and their expression of NKG2D. Without being bound by theory, using a LSRII, which enables multi-parametric testing from a single tube, 2-4 million PBMCs will be more than sufficient for this analysis. This will leave enough PBMCs (>20 million cells) from a 30 ml blood draw to provide for other goals.

Flow Sorting for Downstream Applications Using T Cell Subsets

The goal is to provide potential alopecic T cells for downstream functional analysis. For example, flow sorted T cell subsets will be provided for RNA profiling, for instance, transcriptional profiling of NK-type T cells. For biomarker studies, honing in on the appropriate cellular subset will improve the resolution of the analysis, which will otherwise be diluted by the presence of RNA from the mixed heterogenous PBMC population.

Studies in the AA mouse indicate that the alopecic T cells (FIG. 24 ), are recirculating and can be readily found and isolated from the blood and cutaneous lymph nodes. More than 5 million CD8+ NKG2D+ T cells can be isolated by flow sorting from the cutaneous lymph nodes of a single mouse. This is an impressive number of fresh primary “pathogenic” T cells for downstream study and is a substantial improvement on the cell yields one can expect to recover from “crawl-outs” from alopecic skin. The identification of pathogenic T cell subsets in the blood of AA subjects human AA subjects is a scientific goal. Flow isolated T cell subsets will be provided for downstream applications including biomarker studies, functional studies and spectratyping to provide evidence for immuopathogenic relevance of specific T cells subsets.

i) Spectratype Analysis of TCR Repertoire

Spectratyping or TCR f-chain length distribution analysis of RNA from tissues or phenotypically separated lymphocyte subsets provides a qualitative portrait of the TCR clonotype utilization/repertoire of the infiltrating T cells. In recent years this technique has been used by others (5-7) as a highly sensitive and accurate method to delineate the proportion of clonally expanded T cells in a sample. Oligoclonality, evidence of expansion of subsets of T cells with restricted TCRs is indicative of antigenic drive in inflammatory processes and can be used as a first step to identify pathogenic T cell clones. Since each TCR rearrangement varies in CDR3 length in multiples of three nucleotides, heterogeneity of CDR3 length within a population of T cells can be used as a measure of TCR diversity. The principle of this technique is to PCR amplify the cDNA obtained from a T cell population using an upstream specific Vβ family member primers and a downstream constant region primer that together span the CDR3 region made by combinatorial and junctional VDJ joining. The PCR products are then fluorochrome-labelled in a primer extension (“run-off”) reaction using a fluorescently labeled constant region primer. The products are then run on the ABI PRISM 3700 DNA analyzer. With Gene Mapper software the size of the peaks corresponding to discrete CDR3 lengths can be displayed and analyzed (FIGS. 20 and 27 ). The data are exported and compared to reference polyclonal repertoires, and measures of oligoclonality such as the Hamming distance are calculated. The area of the peaks in each histogram reflects the expression frequency of each CDR3 length within each family. A predominance of clonal expansions in the skin indicates a primary role for dermal antigens in driving disease and will enable focused investigation of pathogenic T cells. In contrast a polyclonal repertoire of unexpanded clones will indicate recruitment by non-clonally specific chemokine receptors. Comparisons of the TCR repertoire in non-lesional skin and blood of the same individuals will indicate whether clonally expanded skin resident T cell populations might also be found in the circulation. Similarly sorted T cell subsets can be used for source RNA material for repertoire analysis. For instance, FIGS. 20 and 27 demonstrates that in C3H mice an oligoclonal population is found in the lesional skin, a repertoire that is also over-represented in NKG2D positive but not negative CD8 lymph node populations. These data confirm that CD8+ NKG2D+ T cells contain most (but not all) of the oligoclonal T cell populations found in the skin. Note that some TCR/clones are found in skin but not in draining cutaneous lymph nodes, e.g., Vβ10. These likely include CD4+ T cell populations found in AA skin but not present in the sorted CD8+LN fraction. This approach provides powerful evidence for “antigen-drive”, and combined with flow sorting as shown here, identifies pathogenic T cell subsets amongst populations of cells.

This evidence of clonal sharing or oligoclonality will be investigated and validated in mouse and human samples at the level of clonotype resolution by preparing bacterial libraries of the PCR products using topoisomerase-based cloning, and sequencing the TCR J-chain of resulting clones in a high throughput manner using 96-well based techniques as described (5-7). Usually 48 or 96 clones are selected and sequenced for each PCR product. The sequences are exported and aligned in GENEIOUS™ and the fine structure of VDJ element usage and joining is determined using Vquest. This allows definitive identification of shared clones and lymphocyte trafficking in different regions, as well as precise enumeration of the clonotypic composition of a sample. Furthermore, clonotypic TCRs can be used for gene transduction studies to generate T cells and retrogenic mice expressing alopecic TCRs. This effort can also lead to development of “humanized” transgenic TCR models of alopecia using transgenically expressed human alopecic TCRs in HLA02 transgenic mice.

ii) Transcriptional Biomarkers of Circulating Immune Cells

A transcriptional signature marking aggressive, drug refractory Lupus can be identified within the CD8 T cell compartment, but not when using RNA of total PBMCs (8) Thus combining flow sorting with transcriptional profiling greatly powers the analytic capacity. In alopecia areata the pathogenic cellular subset in the appears to be CD8⁺ NKG2Dhi T cells that envelop the hair follicle and may be of pathogenic relevance broadly in autoimmunity (9) including both celiac disease (4, 10), Type I diabetes (11) and rheumatoid arthritis (12-14). Transcriptional profiling of these cells in celiac disease (4, 10) previously described an NK-like transcriptional programming of these cells that will be similarly assessed in sort-purified circulating and skin-derived CD8 T cells. It will be important to specifically address the transcriptional profiles of these cellular populations to identify biological pathways central to these cells.

Epigenetically regulated autoimmune genes using ILLUMINA™-based genomic approaches were identified and DNA was obtained from sorted autoimmune human T cells from patients with Type I diabetes, alopecia areata and celiac disease. Hypermethylation of the promoter regions that downregulate the expression of important immunoregulatory genes including CD3, PD-1 and FasL was identified.

iii) Cellular Immunology

Flow cytometryic analysis of activated PBMCs can be used for intracellular cytokine analysis and or signaling activation of phosphorylated proteins. Classic antigen specific, mitogenic or mixed lymphocyte responses can be appreciated with CFSE-stained T cells or with thymidine incorporation. Available equipment includes a cell harvester and Microbeta/Trilux plate reader (for thymidine based proliferation and chromium release cytotoxicity assays), ELISA plate readers/washers, ELISPOT readers.

Skin Immunobiology

Preparation of primary hair follicle and T cell components for functional (cytotoxicity), preparative (T cell cloning) and analytic studies (spectratyping, RNA profiling). Biopsy specimens from AA patients on study are precious, with immunostaining and transcriptional profiling studies taking the highest priority given the feasibility and informative power of these two approaches. Routinely human scalp skin is procured from control individuals (hair transplant donors) to establish primary cultures of individual cellular populations within the skin and hair follicle.

a) Culture of T Cells and Hair Follicle Components, Hair Follicle Organ Culture and Skin Organ Culture.

i) HF targets: Human scalp skin procured from control or AA affected individuals will be used to establish primary cultures of individual cellular populations within the skin and hair follicle (FIG. 28 ) to study the regulation of NKG2DL in primary HF populations and to evaluate HF populations as targets in CTL assays. Interfollicular skin will be dispase treated to separate the epidermal and dermal components and enzymatically processed to establish primary cultures of keratinocytes and fibroblasts. The hair follicles will be microdissected to separate the mesenchymal components and further used to culture dermal sheath cells (DSC) and dermal papilla cells (DPC). Serum free hair follicle organ culture established in the laboratory modeled after protocols from Kondo and Philpott et al (15) (FIG. 30 ) will be used to routinely culture individual follicles from control individuals, as well as lesional (when possible) and control skin from AA patients. The follicles are cultured in serum free growth conditions and show normal anagen growth for 7-10 days, followed by catagen entry and disintegration of the matrix (16). For cytotoxicity assays, the follicles will be transferred to media which sustains cytotoxic T-Cells or NK cells (MYELOCULT™, Stemcell Technologies) for 4-8 hrs. The skin explants maintain normal skin architecture, as well as the immune microenvironment, and will be used for functional studies (17).

ii) T cell effectors: Isolation of sufficient numbers of T cells from enzymatically digested/dispersed human skin can be difficult; however cultured T cells weren expanded using the dermal crawl-out approach developed by Clark (2). Skin explants have been used to expand the resident T-cell population of the skin by culturing the explants on cell foam matrices for 3 weeks in EIDM containing 20% FCS, IL-2 and IL-15. This technique yields 0.3-3.0×106 T cells per 4 mm biopsy enabling immunophenotyping, T cell cloning, Th-profiling, and other downstream applications (e.g., transcriptional profiling, cytotoxicity and other functional assays). For AA patients on intervention trials, if sufficient T cells are obtained after three weeks of expansion (>1×10⁶) they will be divided, with half the population used for RNA isolation/profiling and the other half used for flow immunophenotyping/Th profiling. If the yields are less than 1×106 total cells, studies will be limited to RNA profiling. For AA patients that are not on study, there will be more latitude for using crawl-out T cells and autologous HF targets for alternative functional studies, including for instance cytolyic assays and studies with antibody blockade/small molecules. As an example of the ability to analyse rare clinical populations of T cells, FIG. 29 illustrates a T cell ‘crawl-out” assay, in which cultured human dermis obtained from punch skin biopsies are used as source material for flow cytometric analysis.

Note that T cells obtained from AA skin biopsies are oligoclonal IFN-γQ producing CD8+ NKG2D+ T cells in striking contrast to the expected polyclonal CD4 population seen in crawl-outs from normal skin (2, 18).

b.) Interaction of the Skin and the Immune Components in Alopecia Areata Models

Established protocols are available for assessing skin-immune interactions for the studies herein. Primary cultured cells, as well as organ-cultured follicles, can be used as targets using cytotoxic T cells from control or patient peripheral blood lymphocytes, or those derived from the skin (FIG. 29 ). The immune effector cells can be co-cultured with CFSE-labeled target cells/hair follicles and the cytolysis will be measured colorimetrically by LDH release, or alternatively, the dead cells will be stained with 7-AAD and the cell numbers counted by flow cytometry. These assays can be used to determine molecular requirements for the interaction of immune effectors and HF targets, including cytotoxicity. For instance as shown here, cytotoxicity required NKG2D engagement and prior sensitization of targets with cytokines/TLR ligands known to upregulate NKG2DL transcription and surface expression.

Evaluation of Skin Biomarkers of Pathogenesis: Immunostaining and Transcriptional Profiling

AA shows high correlation with several autoimmune disorders, making the hair follicle a highly accessible organ in which to study basic mechanisms of autoimmunity. There has been substantial interest in identifying early surrogate biological markers of pathogenesis. As such, AA represents a model system for biomarker development which may have relevance to a broad range of diseases. The following biomarkers are currently used to monitor AA development and response to treatment both in humans and in mice.

a) Immunohistochemical Staining for lymphocytic Infiltration. AA is associated with the presence of intrafollicular, and parafollicular immune infiltrate. For immunohistochemistry, tissue will be either fixed in 10% formalin in PBS for 8 hours at room temperature and stored in 70% ethanol for paraffin sections or embedded directly in CRYOMATRIX™ (Shandon, Waltham, Mass.) on dry ice for frozen sections which are stored at −80° C.

Paraffin-embedded tissue blocks are cut into 8-m sections, and hematoxylin and eosin (H&E) staining is carried out for histological studies. Frozen tissue is also sectioned to a thickness of 8 □μm and fixed in 4% paraformaldehyde for immunofluorescence staining. Sections are stained with fluorescence-labeled secondary antibodies and immunofluorescence imaging carried out using Zeiss Axioskop microscope. Basic immunohistological/fluorescent staining will include staining with primary antibodies to CD3, CD4, CD8. As an example of the immunostains, evidence that MHC I and II and ICAM-1 are massively upregulated in alopecic HFs which are associated with a dense CD8 dominated infiltrate is provided (FIG. 31 ).

b) Isolation of total skin RNA and Quantitative PCR for inflammatory markers. The human and mouse skin will be tested for elevation of inflammatory gene transcripts already defined, including IFN-response genes, and gene signatures to be defined, including “NK-type” CD8 T cell transcripts. Total RNA will be extracted from skin using an RNeasy purification kit (Qiagen). DNase-treated total RNA will be reverse-transcribed using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, Calif.).

RT-PCR shall be performed using SYBR™ Green Master Mix and an ABI PRISM 7000™ Sequence Detection System (Applied Biosystems, Foster City, Calif.). GAPDH and -actin will be used as internal normalization control genes.

One of the goals of the studies herein is to establish the phenotype of circulating AA effector populations by matching spectratypes of sorted peripheral blood AA T cell subsets with those found in total skin T cells from the same patient. Flow cytometric sorting techniques will be combined with spectratype analysis to identify pathogenic T cells in the circulation of AA patients. This same approach was used in the mouse model to show that CD8+ NKG2D+ lymph node cells contain the majority of the T cell clones found in alopecic skin. In AA subjects, the TCR repertoire will be compared in lesional and non-lesional skin and blood of the same individuals to identify whether clonally expanded skin resident T cell populations are also be found in the circulation. By progressive refinement of the sort criteria to isolate source RNA material from circulating T cell subsets one should be able to (at least partially) match the TCR repertoire found in total skin from the same patient and in this way identify the immunophenotypic markers of pathogenic CD4 and CD8 T cells. Identification of pathogenic T cell subsets will be enormously valuable for monitoring clinical studies, refining biomarker development and would provide unique cellular materials for bedside-to-bench studies of pathogenesis.

Another goal of the studies herein is to establish the Th profile of AA T cells infiltrating the dermis and circulating in the peripheral blood. Pathogenic AA T cell Effector Differentiation/Cytokine Profile: Therapeutic targeting of specific Th-pathways has succeeded in other human skin inflammatory diseases, most notably in psoriasis a prototypical Th17 disease. There is substantial data in the AA animal model for AA as a predominant Th1 disease (19-23). To establish Th1-predominance in humans and develop the rationale and biomarker platform for Th1-targeted therapies, the Th-profile of infiltrating dermal AA T cells and relevant circulating T cell subsets in the blood will be addressed using a multi-prong approach including multianalyte, RNA profiling, immunostaining and intracellular flow cytometric analysis from the skin and blood. Refining the immunophenotypic markers for AA CD4 and CD8 T cells will of course allow detection of Th-profiles of the potential alopecic-specific T cells amongst the polyclonal circulating population.

Although transcriptional profiling of AA lesions from a small series of subjects are indicative of Th1-type skewing (24), without being bound by theory, AA is not “one disease”. The common occurrence in AA subjects of co-morbid immune conditions that reflect both an underlying Th2 or Th1/17 bias that predisposes to diseases with common pathogenic pathways. including hypersensitivity (25) (dermatitis) and other autoimmune conditions (26-33) (see studies described herein) suggests that there may be a Th2-type AA as well as a Th1 or Th17 type of AA. Thus it is important to identify functional T cell responses in PBMCs from the periphery and from AA lesional tissue explants from a large number of patients that might identify subsets of AA with different Th profiles and to correlate these immune function biomarker parameters with the presence or absence of the relevant SNPs.

The T cell immunophenotype of peripheral T cells and lesional T cells will be assessed. 50 AA patients and 50 normal controls will be studied from the GWAS, with attached clinical history. Assessment of Th-profiles of total circulating T cells is straight forward but multi-parametric flow will be applied to T cell subsets for more granular assessment, as aided ultimately by definition of immunophenotypic markers of AA pathogenic populations. Isolation of sufficient numbers of T cells from enzymatically digested/dispersed human skin can be difficult, and maybe altered by culture conditions. Correlation will be performed with transcriptional profiling using whole skin approaches and microdissected skin samples to describe the cytokine profile within the follicular sheath infiltrate as well as from infiltrates in the interfollicular epithelium.

Human Subjects

Skin biopsies from the scalp will be collected from normal subjects consist of AA patients and unaffected control subjects receiving hair transplantation. Additional biopsies will be obtained from subjects on clinical trials.

The sources of research material will be scalp skin biopsies from AA patients and controls. RNA from small tissue samples, including microdissected hair follicle compartments are routinely obtained. Tissue biopsies from AA patients and controls will be provided.

Vertebrate Animals

Young C3H/HeJ mice (2-3 months) and retired breeders showing visible hair loss will be acquired from Jackson Labs. The mice strains established by each participating principle investigator will be housed in their respective animal housing suites. Mice will be given free access to water and pellet diet (5010 rodent diet, LabDiet, PMI Nutrition International).

Primary cell culture for individual skin and follicle components as well as hair/skin organ cultures will be established for wild-type and transgenic or knock-out mice. These studies also involve hair follicle and skin tissue characterization. The mice will be euthanised by CO2 asphyxiation followed by cervical dislocation, a veterinary-approved and widely recommended method. It is the quickest and most humane method that is not associated with any pain or stress for the animals. Mice will be shaved on the dorsal surface with electric clipper and then skin tissue will be dissected out. The isolation scheme of individual cellular components of the skin and hair follicle as well the T-cells isolation from skin explants is described in FIGS. 25, 28 and 29 . Skin Samples will also be embedded in OCT and paraffin. In addition to skin, spleen and thymus will also be dissected from the animals and further used for flow cytometry. Serum from the animals will also be collected by tail bleeding and used for cytokine profiling.

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Example 6

In vivo pre-clinical studies of blocking Jak1/Jak2 was performed using the INCB018424 Jak1/Jak2 inhibitor in the grafted animal model of the disease, C3H-HeJ mice. 0/5 mice developed alopecia after treatment with INCB018424, whereas 2/5 placebo treated mice developed AA.

Example 7—Mouse Transplant Model for Alopecia Areata

Alopecia areata (AA) is one of the most prevalent autoimmune diseases and manifests as nonscarring hair loss. The course of the disease is unpredictable, and there are currently no consistently efficacious treatments available. Although research into disease pathogenesis and the development of targeted therapies are lacking, the creation of a mouse transplant model with a high incidence of disease has opened up new avenues of preclinical experimentation.

Several lines of evidence support the potential efficacy of JAK3 inhibitors in the treatment of AA: (1), a recently completed genome-wide association study of AA identified a number of gene-associated loci with immunological relevance, including those for IL-2/IL-2RA and IL-21; (2) initial findings in a preclinical mouse model suggested disruption of IL-15/IL-15R signaling ameliorates the disease; and (3) data indicate that T cells, dependent on IL-7 and IL-15 for survival, mediate the disease in the mouse model and in humans.

We therefore assessed the efficacy of JAK3 inhibition on the development of alopecia in a preclinical mouse model. Alopecic C3H/HeJ skin grafts were transplanted onto unaffected C3H/HeJ mouse recipients and subsequently treated with a JAK3 inhibitor administered by osmotic pump, vehicle administered by osmotic pump, or PBS injections. While 4/4 PBS-treated and 2/2 vehicle-pump treated mice developed alopecia by six weeks following transplantation, 0/5 mice treated with the JAK3 inhibitor developed alopecia. Microarray analysis of skin, immunohistochemical stains of skin for T cell infiltrate, and serum marker assessments demonstrated decreased inflammatory profiles in mice treated with JAK3 inhibitors. In total, our findings suggest JAK3 inhibitors may be an effective treatment in patients with AA.

Example 8—Targeting the Immune Effector Response in AA

Specific autoimmune mechanisms underlying Alopecia Areata (AA) have remained obscure and therefore clinical investigation of AA has historically lagged behind other autoimmune diseases.

Identify Effective, Clinically Relevant, Therapies in Mouse Models of Alopecia Areata.

In both human AA and in the AA mouse model, local hair follicle (HF) IL-15/NKG2DL upregulation was identified as key inflammatory signals that recruit/activate CD8⁺ T cells, likely the critical AA immune effectors responsible for IFN-gamma (γ) production and hair follicle (HF) cytotoxicity. These observations provided the rationale for the studies to therapeutically target the IL-15/NKG2D.

It has been shown that the IL-15 pathway can be blocked using JAK3 protein tyrosine kinase inhibitors (PTKi).

Small molecule PTKi prevents AA. 5 mice were treated systemically with tofacitinib (CP-69055; 10 mg/kg/day within an Alzet pump). Tofacitinib is a JAK3 inhibitor known to inhibit IL-15 signaling in human whole blood assays. None of the treated mice developed alopecia areata or cutaneous lymphadenopathy, whereas untreated mice manifested both AA and associated cutaneous lymphadenopathy (FIG. 32 ). Topical delivery approaches will be pursued for AA reversal in the mouse model that has the major benefit of an improved therapeutic index for clinical development.

Identify Disease-Associated AA Biomarkers and their Reversal with Effective Therapy in C3H Mice.

Cellular, inflammatory, and molecular biomarkers have been assessed in treated mice. Targeted therapy with tofacitinib eliminated the pathogenic CD8⁺ NKG2D^(hi) cells from both the skin and the cutaneous draining lymph nodes and moreover down-regulated inflammatory biomarkers in the skin, including MHC molecules and the AA-associated IFN signature (FIG. 33 ). HF NKG2DL expression was reduced although not completely eliminated (e.g. H60 expression by immunostain) (FIGS. 33 and 34 ), indicating H60 upregulation as a proximal event.

Transcriptional profiling analysis of treated vs. untreated skin shows abrogation of the cutaneous IFN-signature with treatment. These murine transcriptional profiles will be integrated computationally with transcriptional profiles obtained from human skin biopsies from untreated alopecic subjects to identify candidate dynamic human biomarkers that are associated with patchy stable disease vs. progressive disease vs. alopecia universalis.

Interpretation of PTKi targeted approaches. PTKi's vary greatly in their selectivity of their target kinases. For tofacitinib, JAK sensitivity is greater for JAK3>JAK1>>JAK2 (IC₅₀ is 28-50 nM, 140-180 nM, 1000-5000 nM, in whole blood assays, respectively). In the treated mice, tofacitinib serum levels would be 30-40 nM, and potential impacts will be assessed on both JAK1 and 3 activity. For ruxolitinib (a JAK1/2 inhibitor), sensitivity is JAK2>JAK1>>JAK3 (IC₅₀ is 3.3 nM, 2.8 nM, and 428 nM for in vitro kinase inhibition, respectively). Pharmacodynamic JAK inhibition in the mouse is likely to be transient since ruxolitinib dosing is once daily and the half-life in rodents is 3-6 hours.

For example, a Jak3 inhibitor can exert selective inhibition activity of its JAK3 target at about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, or at about 55 nM. A Jak3 inhibitor can also exert selective inhibition activity of its JAK3 target at about 60 nM, about 65 nM, about 70 nM, about 75 nM, about 80 nM, about 85 nM, about 90 nM, about 95 nM, about 100 nM, about 105 nM, about 110 nM, about 115 nM, about 120 nM, about 125 nM, about 130 nM, or at about 135 nM.

For example, a Jak3 inhibitor can also exert inhibition activity of its JAK3 target, while also able to promiscuously exert inhibition activity of a JAK1 target, at about 140 nM, about 145 nM, about 150 nM, about 155 nM, about 160 nM, about 165 nM, about 170 nM, about 175 nM, about 180 nM, about 185 nM, about 190 nM, about 195 nM, about 200 nM, about 205 nM, about 210 nM, about 215 nM, about 220 nM, or at about 225 nM.

For example, a Jak3 inhibitor can also exert inhibition activity of its JAK3 target at about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about 650 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, or at about 950 nM.

For example, a Jak3 inhibitor can also exert inhibition activity of its JAK3 target, while also able to promiscuously exert inhibition activity of a JAK2 target, at about 1000 nM, about 1250 nM, about 1500 nM, about 1750 nM, about 2000 nM, about 2250 nM, about 2500 nM, about 2750 nM, about 3000 nM, about 3250 nM, about 3500 nM, about 3750 nM, about 4000 nM, about 4250 nM, about 4500 nM, about 4750 nM, about 5000 nM, about 5250 nM, about 5500 nM, about 5750 nM, about 6000 nM, about 6250 nM, about 6500 nM, about 6750 nM, about 7000 nM, about 7250 nM, about 7500 nM, about 7750 nM, or at about 8000 nM.

For example, a Jak1/2 inhibitor can exert selective inhibition activity of its JAK1 and/or JAK2 target at about 0.5 nM, about 1.0 nM, about 1.5 nM, about 2.0 nM, about 2.5 nM, about 3.0 nM, about 3.5 nM, about 4.0 nM, about 4.5 nM, about 5.0 nM, about 5.5 nM, 6.0 nM, about 6.5 nM, about 7.0 nM, about 7.5 nM, about 8.0 nM, about 8.5 nM, about 9.0 nM, about 9.5 nM, or about 10.0 nM. For example, a Jak1/2 inhibitor can exert selective inhibition activity of its JAK1 and/or JAK2 target at about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 75 nM, about 80 nM, about 85 nM, about 90 nM, about 95 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, or at about 400 nM.

For example, a Jak1/2 inhibitor can also exert inhibition activity of its JAK1 and/or JAK2 target, while also able to promiscuously exert inhibition activity of a JAK3 target, at about 425 nM, about 450 nM, about 475 nM, about 500 nM, about 525 nM, about 550 nM, about 575 nM, about 600 nM, about 625 nM, about 650 nM, about 675 nM, about 700 nM, about 725 nM, about 750 nM, about 775 nM, about 800 nM, about 825 nM, about 850 nM, about 875 nM, about 900 nM, about 925 nM, about 950 nM, about 975 nM, or at about 1000 nM.

To activate a Jak protein target in the skin, the skin should be penetrated at about 0.5 mm, about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm, about 3 mm, about 3.5 mm, or at about 4 mm. Depending on the depth of penetration, the concentration of a JAK inhibitor, such as a JAK1/2 inhibitor or a JAK3 inhibitor, can range from about 25 nM to about 5000 nM. At the higher concentration ranges, one JAK inhibitor can elicit an inhibitory effect not only of its target but also of other JAK proteins.

Integration of Human and Mouse Gene Expression Profiles.

The ribbon plots shown in FIG. 35 depict microarray data of normalized skin expression profiles of select genes from mice treated with 13 weeks of the JAK3 inhibitor CP690550 and PBS control. Briefly, after 13 weeks of treatment with the JAK3 inhibitor, skin was harvested and subjected to microarray analysis and compared with that of mice treated with PBS. The data show treatment-related elimination of the IFN-response chemokines and markers of CD8 cytotoxic effectors. Expression arrays from mouse and human will be integrated to compare expression profiles and eventually response to treatment.

Example 9—Systemic Treatment with JAK3i

“NK-type” CD8αβ+ T cells are massively expanded in alopecic skin and draining LNs, and are required for T cell mediated transfer implicating this cytotoxic cell subset as the likely pathogenic effectors.

IL-15 is a key cytokine responsible for inducing CD8 T effectors in vitro. Moreover, IL-15 is produced by intestinal epithelial cells and is a known precipitant of CD8 cytotoxicity in celiac disease.

Alopecia areata is marked by IL-15/IL-15Ra upregulation in the human and murine hair follicle and implicates this cytokine as an end-organ trigger of CD8-mediated autoreactivity in AA.

Systemic IL-15 blockade with a small molecule JAK3 PTKi effectively prevented alopecia areata, eliminating expansion of the pathogenic NK-type CD8 population and ablating the inflammatory signature in the skin. FIG. 53 shows reversal of established disease with topical therapies.

Example 10—Cellular Identity of Cytotoxic T Lymphocytes in Alopecia Areata Defines a Therapeutic Strategy

Alopecia areata (AA) is a common autoimmune disease resulting in an immune attack on the hair follicle¹. Although T cells have been implicated in the disease process, the pathways underlying their activation had not been determined². Prior to the GWAS study, the genetic basis of AA was largely undefined. Unexpectedly, a region of strong association was identified within the ULBP gene cluster on chromosome 6q25.1, encoding activating ligands of the natural killer cell receptor, NKG2D, which had never before been implicated in an autoimmune disease. Guided by the GWAS studies implicating NKG2D ligands (NKG2DL)³, here ‘NK-type’ CD8+ T cells were identified as the dominant effectors, which are both necessary and sufficient for disease induction. Global transcriptional profiling of mouse and human AA skin revealed striking signatures indicative of a robust IFNγ response and the presence of a cytotoxic T cell infiltrate.

Using the graft model of C3H/HeJ mouse skin to transfer AA, disease prevention can be recapitulated when treating at the time of grafting, as well as reversal of established disease by allowing grafted mice to first lose their hair. Systemically-administered pharmacological inhibitors of the JAK3 protein tyrosine kinases eliminated the IFN-signature and prevented the development of AA, and topical administration reversed established disease. Notably, these effects were durable up to 3 months after cessation of therapy. These findings illustrate the power of GWAS studies in uncovering new disease mechanisms, which have rapidly translated into new therapeutic opportunities in AA.

AA is a T cell mediated autoimmune disease characterized by hair loss and, histologically, by infiltrating T cells surrounding the hair follicle bulb, the so-called “swarm of bees^(1,2). Previous studies have shown that transfer of total T cells (but not B cells or sera), can confer the disease in human xenograft models as well as the C3H/HeJ mouse model^(4,5). However, the identity of the specific pathogenic T cell subsets in either human or mouse AA has not yet been defined.

Guided by a previous GWAS study, which identified ULBP3/6 as the most highly significant non-HLA risk locus in AA (p=2×10⁻²⁰)³, a focus has been centered on establishing NKG2DLs as potential “danger signals” in the disease process. Upregulation of ULBP3 has previously shown in human AA hair follicles, associated with a dense infiltrate of CD8⁺ NKG2D⁺ T cells³. Here, the role of these cells in AA immunopathogenesis, as well as a target for potential therapeutic intervention, was investigated.

To determine the mechanistic importance of these cells in pathogenesis, the C3H/HeJ mouse model of AA was used, in which spontaneous disease develops with ˜15% incidence between 6 and 12 months of age⁶, and the graft-transfer C3H model was used, in which grafts from affected animals can transfer disease to 100% of C3H/HeJ recipients in 8-12 weeks time⁷. Lesional skin biopsies revealed that CD8⁺ NKG2D⁺ T cells infiltrate the epithelial layers of the hair follicle that overexpress the NKG2DLs H60 and Rae-1, analogous to the situation in human AA (FIG. 54A-B and FIG. 58A-B). Flow cytometric examination of the CD45⁺ leukocyte population in the skin identified a striking expansion of CD8⁺ NKG2D⁺ T cells (FIG. 54D). Other cell types, including CD4⁺ T cells were present in much smaller numbers (FIG. 58C). C3H/HeJ mice exhibit striking cutaneous lymphadenopathy (FIG. 54C) with increased total cellularity and, as noted in the skin, a dramatic expansion of a CD8⁺ NKG2D⁺ cell population not present in disease-free C3H/HeJ mice (FIG. 54C-D).

The immunophenotype of the skin-infiltrating CD8+ T cells were similar to the CD8⁺ NKG2D+ population found in the cutaneous lymph nodes: CD8ab⁺ effector memory T cells (CD8^(hi)CD44^(hi)CD62L^(low) CD103⁺) bearing several NK immunoreceptors, including CD49b and NKG2A/C/E (FIG. 54E and FIG. 58D). These ‘NK-type’ CD8+ T cells expressed high levels of IFNg and exhibited NKG2D-dependent cytotoxicity against ex vivo expanded syngeneic dermal sheath target cells (FIG. 54F). Gene expression analysis of the CD8⁺ NKG2D⁺ T cells isolated from alopecic C3H/HeJ lymph node cells using RNAseq demonstrated a transcriptional profile characteristic of effector CTLs as defined by the ImmGen Consortium⁸⁻¹⁰ and identified several additional “NK-specific” transcripts (FIG. 54G and FIG. 59 and Table 14).

TABLE 14 Differentially expressed genes in CD8+NKG2D+ memory T cells vs. CD8⁺NKG2D⁻ memory T cells isolated from cutaneous lymph nodes obtained from alopecic mice. RNAseq was performed on cRNA made isolated from flow-sorted cells (BD influx) from total cutaneous lymph nodes from two alopecic mice. GeneSymbol FC Gstm5 63.99 Gzma 52.13 Ifitm1 44.81 Cx3cr1 38.24 Sprn 35.20 Tjp1 34.76 Igsf5 32.13 Tmem132e 31.71 Cmklr1 30.91 5430421N21Rik 30.69 Csf1 29.97 Olfr60 28.79 4933431E20Rik 27.87 Metrnl 27.44 Epdr1 27.43 Cd244 27.22 Gpr141 26.45 Ccr1 26.17 Ccr8 23.75 Pkd2 23.23 Lgals3 22.66 Esm1 21.59 AI661453 20.69 Sema6d 20.58 Tmem171 19.13 Havcr2 19.01 Rasgrf1 18.32 Bean1 18.25 Adora3 18.13 Itga1 17.66 Tpbg 17.52 Gzmb 17.43 Gpr44 17.11 Stk32c 16.81 Bcar1 16.72 Cntn1 16.70 Slc38a8 16.65 Adam8 16.15 Usp44 15.85 B4galnt4 15.50 Cass4 14.94 Galr3 14.86 Ifitm2 14.77 Rimbp2 14.64 Fam171b 14.02 Srxn1 13.78 Ptger3 13.70 Lrat 13.48 Trpm6 13.43 Lrrn2 13.17 Arnt2 13.06 Yap1 13.04 Anxa1 12.85 Gpr56 12.78 Rai14 12.59 Lypd3 12.52 Gm5127 12.23 Pdgfrb 12.13 Tktl1 11.87 Gzmk 11.63 Olfr525 11.61 AF529169 11.57 Olfr523 11.48 Selm 11.43 Sema4c 11.25 Ppp2r2c 11.12 Fxyd4 11.09 Sept5 11.07 Styk1 11.04 Myadm 10.84 Wnt10b 10.65 Fgl2 10.61 Gcnt4 10.59 Emp1 10.44 Cacng8 10.38 Car5b 10.36 Ly6g5b 10.32 Lmna 10.16 Vipr2 10.08 Ptpn5 10.01 Plekhf1 9.92 Il13 9.87 9430020K01Rik 9.87 Klrk1 9.80 Itsn1 9.65 Ret 9.62 Itgae 9.58 Col6a3 9.50 Nbea 9.41 Rab27b 9.41 Clec12a 9.36 Mgat3 9.35 Zfp57 9.30 S100a4 9.27 AI414108 9.22 Trim58 9.19 Irak3 9.18 Gpr34 9.15 Mt3 9.04 AW555464 9.02 Perp 9.00 Ace 8.94 Tmeff2 8.92 Slc35f3 8.81 Dyrk3 8.81 Nxnl2 8.72 Galnt3 8.67 Cish 8.63 Vash1 8.58 Klre1 8.58 Dmrt2 8.56 Wnt9a 8.48 Dusp14 8.43 Dmd 8.29 St6galnac2 8.26 Slc41a2 8.26 Cdh1 8.22 Tead1 8.09 Msc 8.05 Ttc39c 8.01 Slc27a6 7.94 Hlx 7.85 Mid2 7.73 Plod2 7.70 Prkcc 7.68 Ptgs1 7.63 6430571L13Rik 7.60 Specc1 7.55 Prss16 7.53 Fam129b 7.49 Spns3 7.49 Lonrf3 7.45 Fat4 7.41 Nphs2 7.39 Cpd 7.34 Cdkn2a 7.26 Il17rd 7.14 Nebl 7.12 Src 7.10 Scn1b 7.07 Sccpdh 7.04 Igsf9b 7.03 Crispld2 7.02 Plscr4 7.01 Tmigd1 6.96 Camk2n1 6.95 Trat1 6.87 Mtap6 6.85 Qpct 6.85 Thsd7b 6.82 Tceal3 6.76 Nlrp12 6.75 2010002N04Rik 6.70 Lat2 6.68 Susd2 6.61 Ptprj 6.61 P2ry14 6.56 Csgalnact1 6.55 Klri2 6.54 1700001C19Rik 6.48 Spp1 6.43 Hlf 6.41 Gpx8 6.39 a 6.37 Rtn4r 6.31 Lamc1 6.25 Kndc1 6.23 Slc22a15 6.19 Id2 6.15 Tnfsf13b 6.14 Elovl4 6.13 5830411N06Rik 6.04 Plxnd1 6.00 Vstm2a 5.98 Rora 5.96 Il2 5.93 Klrc1 5.92 Tesc 5.86 Cd40lg 5.84 Glis2 5.84 Mt1 5.83 Mdfic 5.82 P2ry2 5.73 Hpse 5.72 Ttc16 5.70 Kitl 5.66 Arhgef25 5.65 Foxf2 5.62 Kir3dl2 5.56 Unc79 5.52 Aim1l 5.47 Rgag4 5.44 Pygl 5.41 Efcab6 5.39 Htra3 5.39 Card10 5.39 Nek6 5.38 6720401G13Rik 5.36 Fam131b 5.36 Grb14 5.34 Pde1c 5.33 Pld1 5.31 Gipc2 5.30 8430427H17Rik 5.30 Gabarapl1 5.29 Olfr527 5.28 Tenc1 5.27 Snx7 5.27 Rgs8 5.25 Slc16a7 5.24 Alcam 5.23 Tmbim1 5.23 Nefl 5.22 Pdcd1 5.21 2810030E01Rik 5.21 Abhd3 5.19 Clnk 5.18 Nlrp1b 5.14 Cav2 5.13 Aff2 5.12 Kbtbd13 5.10 Ppap2a 5.07 Gm14718 5.06 Pde4a 5.03 Cnga2 4.95 Acvrl1 4.92 Mapkapk3 4.92 Ccr2 4.87 Hip1 4.84 Pkp2 4.83 Nap1l3 4.83 Gorasp1 4.82 Zfp651 4.82 Hmga2-ps1 4.81 Tceal1 4.81 Atp1a2 4.81 2200002K05Rik 4.81 Mmp2 4.77 Ankrd35 4.77 Klrc2 4.77 Ptgfrn 4.74 Ildr1 4.66 Ptpn3 4.64 5031425F14Rik 4.62 2510009E07Rik 4.58 Emilin2 4.58 2900026A02Rik 4.57 Cd101 4.54 9030425E11Rik 4.52 1500009L16Rik 4.50 Cdh17 4.48 Clec5a 4.48 Dnahc9 4.47 Gpr25 4.45 Ifng 4.44 Socs2 4.42 Pard3 4.42 Nhsl2 4.41 Layn 4.40 Csf2 4.35 Crybb3 4.30 Cxcr6 4.30 Zeb2 4.27 Prkar2b 4.27 Fam70a 4.26 Gdpd5 4.23 Gm8773 4.22 Fbn1 4.20 Itgax 4.20 Rbpj 4.17 Cxcl13 4.15 Tmem2 4.14 Tmem205 4.14 Klrc3 4.13 Gcnt1 4.13 Zc3h12c 4.12 Mir1199 4.09 Ermn 4.08 Agap1 4.07 1810033B17Rik 4.07 Klf8 4.06 4831426I19Rik 4.06 Ninl 4.06 Fbxo44 4.04 Rab39b 4.03 Tspan2 4.02 Adrb1 4.00 Spns2 3.98 Osbpl3 3.97 Rapsn 3.96 Dhrs9 3.94 Mboat2 3.90 Pparg 3.90 Clip4 3.89 Apol9a 3.88 Marveld2 3.86 Capn5 3.86 Ranbp17 3.86 Ncam1 3.85 Ctnnd2 3.85 Slc25a24 3.85 Palm 3.84 Fcer1g 3.79 Gzmc 3.78 Prdm1 3.78 Scn8a 3.75 Fkbp9 3.72 Gstt1 3.71 Adssl1 3.71 Prrg4 3.71 Kir3dl1 3.70 Ssbp2 3.67 Swap70 3.67 Trp73 3.67 Il12rb2 3.66 Phka1 3.66 Xcr1 3.65 Stau2 3.64 9130019P16Rik 3.63 Clec7a 3.63 6430531B16Rik 3.63 Marco 3.62 Sulf2 3.61 Reep1 3.61 Anxa4 3.61 Procr 3.60 Slc40a1 3.60 Tnfrsf8 3.59 Matk 3.56 Cysltr2 3.56 Smtn 3.55 Rasl11b 3.54 Amph 3.52 Cysltr1 3.52 Klri1 3.52 B4galt2 3.51 Mlf1 3.49 B4galt4 3.49 Slc4a11 3.47 Htra1 3.47 Ebf1 3.44 Mpp2 3.44 Ica1 3.44 Tigit 3.43 Lzts1 3.43 Epha3 3.43 Mrgpre 3.43 Vdr 3.42 Npnt 3.41 D630039A03Rik 3.40 Rapgef3 3.37 Cnksr1 3.37 Napsa 3.37 E030011O05Rik 3.37 1700019E19Rik 3.37 Ncald 3.37 Ccl8 3.36 Dhrs3 3.36 Ppap2b 3.36 Gas2l1 3.36 St3gal2 3.35 Phactr2 3.34 Rasd1 3.34 Mafa 3.33 Olfr433 3.33 L1cam 3.31 Pik3r6 3.31 C1qa 3.31 Fgd1 3.30 Gm17745 3.30 Apol9b 3.30 Cmtm7 3.29 Bicd1 3.29 Rgs2 3.29 Nos1ap 3.29 Bcl2a1d 3.29 Eif4e3 3.28 Sgtb 3.28 Fut7 3.28 Kif19a 3.27 Mtmr7 3.27 Fam114a1 3.27 Sdc4 3.27 Farp1 3.26 Fbxl21 3.25 Ccrl2 3.25 H1fx 3.24 Eif2c4 3.24 Ube2l6 3.23 Gpr68 3.23 4930544D05Rik 3.23 Hrh4 3.22 Ryr1 3.22 9030418K01Rik 3.22 Acot11 3.22 Pon3 3.22 Serpine2 3.20 Ly6a 3.20 Lrrn3 3.19 B3galt5 3.19 Ankrd29 3.19 Ccl1 3.19 Serpina3f 3.19 St14 3.15 Dscam 3.14 Fam129a 3.14 Med12l 3.14 1300014I06Rik 3.13 Rnf216 3.13 St3gal5 3.13 Slc35e4 3.12 Rab3il1 3.12 Tmem176a 3.12 Syngr3 3.12 Il9r 3.12 Lrrk2 3.11 Zfp532 3.11 Ache 3.10 Wipf3 3.09 Cdkn1a 3.09 Ccdc136 3.08 Epn2 3.07 Fam167a 3.07 Syp 3.07 Spry2 3.06 Tiam1 3.06 Gnaq 3.05 Armcx6 3.05 Raph1 3.05 F730043M19Rik 3.05 Lag3 3.05 Wscd1 3.04 Dkkl1 3.04 Zfp839 3.04 Armcx1 3.03 Zkscan16 3.03 1700009P17Rik 3.02 Nkd1 3.01 Cdkn2b 3.01 Igfbp6 3.01 Dlg2 3.01 Plscr1 3.00 Cacna1i 3.00 Ckm 3.00 Kcnmb4 3.00 Arhgap8 2.99 Gspt2 2.99 Foxd2 2.98 Vat1l 2.98 Bfsp2 2.98 Prrt2 2.98 Wbscr17 2.98 Car13 2.98 Muc20 2.97 Bzrap1 2.96 Frem2 2.96 Fcer2a 2.96 Chn2 2.95 Blk 2.95 Kcnk5 2.95 Mpzl1 2.95 Aplp1 2.95 Tlr4 2.95 H2-DMb2 2.95 Lyn 2.94 Meis2 2.94 Camk1 2.93 Pacsin3 2.93 Tub 2.93 Cebpd 2.93 Gm5111 2.92 Fasl 2.92 Lpcat2 2.91 Mybpc2 2.90 Sytl3 2.90 H2-Eb2 2.90 Hdac11 2.89 Slc43a3 2.89 Klf12 2.89 Blnk 2.89 Atp2b4 2.88 Faim3 2.88 C1qc 2.88 Epb4.1l3 2.87 Ms4a1 2.87 Prr5l 2.86 Fcrla 2.86 Ell3 2.86 Prdx4 2.85 Adcy9 2.85 Pbx3 2.85 Chmp4c 2.84 Osbpl1a 2.84 Apbb1 2.84 Cd79a 2.84 Slc6a8 2.83 Zcchc14 2.83 5730416F02Rik 2.83 Gm9199 2.82 Ern1 2.82 Lxn 2.82 Armc2 2.82 Cr2 2.82 Ptpn13 2.81 Bcl11a 2.81 Scd1 2.81 Chst3 2.80 Gcnt2 2.80 Lyl1 2.80 Itga2 2.79 Scn4a 2.78 Ccl5 2.78 March1 2.76 Siglecg 2.76 Islr 2.76 Spib 2.76 Ndrg1 2.75 Tex15 2.75 Rgs1 2.74 5031414D18Rik 2.74 Frmd4b 2.74 Ankrd6 2.74 Bcar3 2.74 Rhbdf1 2.72 Slco4a1 2.72 Capg 2.71 H2-DMb1 2.71 Hhex 2.71 Postn 2.71 Myo1e 2.71 Il15 2.70 Soat2 2.69 Casp1 2.69 Itga3 2.69 Rtn1 2.68 Sema6b 2.68 Ciita 2.68 Haao 2.68 Atf6 2.68 Kcng1 2.67 Ptk7 2.67 Kdelc2 2.67 Cd180 2.67 Rhbdl3 2.66 Cd86 2.66 Naip5 2.66 Slc15a3 2.65 BC013712 2.65 Cd74 2.64 C77080 2.64 Fam43a 2.63 S100a6 2.63 Lmo2 2.63 Col8a2 2.63 Celf5 2.63 Lhfpl4 2.63 Col1a1 2.62 Ppap2c 2.62 Ccl3 2.62 Rasgrp3 2.62 Klhl30 2.62 Clu 2.62 Cd19 2.61 Syk 2.61 Cpne7 2.61 Rasgef1b 2.61 Gm11435 2.60 Vpreb3 2.60 Lhx2 2.58 Cybb 2.58 Mapk8ip1 2.58 Hck 2.57 1110046J04Rik 2.57 Fam59a 2.57 Stx11 2.56 Btk 2.56 H1f0 2.56 Vav3 2.56 F2rl2 2.55 Hspa2 2.55 Slc29a4 2.55 Tubb4a 2.54 Arsb 2.54 Trim7 2.54 Mef2c 2.54 Asb14 2.54 2010001M09Rik 2.54 Ehd2 2.54 Itpripl2 2.53 Ifitm3 2.53 Zfp941 2.53 Pax5 2.53 Cd24a 2.52 Arhgef40 2.51 Rab30 2.51 H2-Eb1 2.50 Serpinb6a 2.50 Kynu 2.50 Klra7 2.48 Maf 2.48 Fcgr2b 2.48 H2-Ea-ps 2.47 Crip3 2.47 Abhd4 2.47 Fcrl1 2.47 Egln3 2.47 Kif15 2.46 H2-Ab1 2.46 Jazf1 2.46 Dennd5a 2.46 Hgfac 2.45 Rtp3 2.45 9130206I24Rik 2.45 Cited2 2.45 Sult4a1 2.45 Gls2 2.44 Cd80 2.44 Tmem176b 2.44 Vcam1 2.44 Ptms 2.43 Tmem154 2.43 Ccl4 2.43 Rgs16 2.42 Pawr 2.42 Wdfy4 2.42 Olfm1 2.42 H2-Aa 2.42 Stac2 2.41 IL18 2.40 Fam164a 2.40 Phlda3 2.39 Ctnnal1 2.39 Tnfsf10 2.39 Cubn 2.38 Abi2 2.38 2410004P03Rik 2.38 Trim36 2.38 Bmf 2.37 Adap1 2.36 Rarg 2.36 Tmem151a 2.36 Ly6d 2.36 Irf5 2.35 Bhlhe40 2.35 Zfp608 2.34 Fam184a 2.33 Mafb 2.33 Coch 2.32 Gpr179 2.32 Ffar1 2.32 H2-Ob 2.32 Nav2 2.32 Srpk3 2.31 Cd209b 2.31 Arhgef12 2.31 Arvcf 2.31 Serpina3g 2.30 Ppp1r3f 2.30 Cdc25c 2.30 Tmem159 2.30 Atcay 2.30 Sept4 2.29 Cacna1e 2.29 Plxna1 2.29 Fzd6 2.29 Zcchc24 2.29 Ttbk1 2.28 Cd40 2.28 Atp6v0a1 2.28 Gnao1 2.27 Gsg2 2.27 Dock5 2.26 Tlr9 2.26 Fbxo6 2.26 2410022L05Rik 2.26 Ltbp4 2.25 Ncf2 2.25 Cela1 2.25 Ncr1 2.25 Irf4 2.25 Bank1 2.24 Ell2 2.23 1190002F15Rik 2.23 Unc5b 2.23 Bspry 2.23 D17H6S56E-3 2.23 Chst2 2.23 Slc41a3 2.22 Ctla2b 2.22 Gas2l3 2.22 Casp4 2.22 Dcxr 2.21 Ndrg4 2.21 Muc1 2.21 Lass6 2.21 Pls3 2.21 Dok3 2.20 Carhsp1 2.20 Ppfibp1 2.20 Stxbp1 2.20 Ctsh 2.20 Edaradd 2.20 Galnt10 2.20 Prr5 2.19 Rcbtb2 2.19 Pik3c2b 2.19 Nqo2 2.19 Fbxo30 2.18 Mki67 2.17 Prnp 2.17 Ahr 2.16 C030034L19Rik 2.16 Lgals1 2.16 Gm11110 2.15 Jup 2.15 Fam20a 2.15 Sfpi1 2.15 Lrfn4 2.14 Cdkn2c 2.14 Crybg3 2.14 Trerf1 2.14 Dok4 2.14 Cdk6 2.13 S1pr3 2.13 Rxra 2.13 Cd163l1 2.12 Pwwp2b 2.12 1700017B05Rik 2.12 2310010M20Rik 2.12 Nek2 2.12 Lix1l 2.12 Ggta1 2.11 Rorc 2.11 Fosb 2.11 Rimkla 2.11 Txlnb 2.10 Smox 2.10 Tcf4 2.10 Fgd2 2.10 Cd300lf 2.10 Map3k13 2.10 Neil3 2.09 Optn 2.09 A930038C07Rik 2.09 Klra5 2.08 Cyp4f18 2.08 Akap17b 2.08 2700081O15Rik 2.07 Pcyox1l 2.07 Cd226 2.07 Aurkb 2.07 St6galnac6 2.06 Mfge8 2.06 Slamf1 2.06 Zdhhc2 2.06 Bag2 2.05 Gpr55 2.05 Dip2a 2.05 BC064078 2.05 S1pr5 2.05 Mlkl 2.04 Arap3 2.04 Ctla2a 2.04 Gypc 2.04 Ccdc88a 2.04 Serpinb1a 2.03 Fam19a3 2.03 Cdk1 2.03 Trib1 2.01 Gadd45b 2.01 Pfn2 2.01 Klrd1 2.01 Trp53i11 2.01 Cd22 2.00 Mtss1 2.00 Adam19 2.00 Nusap1 2.00 Hk3 0.50 Pak1 0.50 Irgc1 0.50 Zfp296 0.50 Il6st 0.50 Oas2 0.49 Il6ra 0.49 Ctsf 0.49 Lef1 0.49 Gm4951 0.49 Gpr133 0.49 Olfr1033 0.49 Bambi-ps1 0.48 Gtf2ird1 0.48 Dtx1 0.48 Maml3 0.47 Plaur 0.47 Id3 0.47 Tnfrsf25 0.47 Susd4 0.47 Wfikkn2 0.47 4921525O09Rik 0.46 Klra3 0.46 1110032F04Rik 0.46 Car12 0.46 Pard6g 0.46 Slc7a4 0.46 Lrp12 0.46 Spats2l 0.45 Dleu7 0.45 Cacna2d4 0.45 Dst 0.45 Osbpl6 0.45 Tmem108 0.44 4930417O13Rik 0.44 Angptl2 0.44 Adam6b 0.44 Lrrc9 0.44 2310042E22Rik 0.44 Lars2 0.44 Ankrd55 0.44 Ly6k 0.43 Kazn 0.43 Dapl1 0.43 Slc16a5 0.43 Unc13a 0.42 Gpr113 0.42 Siglech 0.42 Olig3 0.42 Ksr2 0.42 Card9 0.42 Rasip1 0.42 Dlc1 0.41 Gm19705 0.41 Syde2 0.41 Actn1 0.41 Epx 0.41 Mir5109 0.41 Gpr83 0.40 Neb 0.40 Sall2 0.40 Fam5b 0.40 Prickle1 0.40 Nme4 0.40 Lif 0.40 Auts2 0.39 Col6a4 0.39 Ccdc164 0.39 Apol11b 0.39 Plac8 0.39 Kcnf1 0.38 Gm15708 0.38 Wnt5b 0.38 Plat 0.38 Nmnat2 0.38 4933440M02Rik 0.38 Dcaf12l1 0.38 Nacc2 0.38 She 0.37 Snord69 0.37 Mical3 0.37 Cox6a2 0.36 Dmbt1 0.36 Aldh1l1 0.36 Scarna6 0.36 Lrp3 0.35 Car11 0.35 Il1r2 0.35 Atp6v1g3 0.35 Clec9a 0.34 Slc6a9 0.34 Tnni3 0.34 Gpr125 0.34 Ikzf2 0.34 Slc7a10 0.33 Lman1l 0.33 A430105I19Rik 0.33 Eng 0.32 Snora17 0.32 Gria4 0.32 Fgfr1 0.32 BC048644 0.31 D830046C22Rik 0.31 Hs6st2 0.31 Hoxa5 0.31 Alpl 0.30 Pgpep1l 0.30 Adam6a 0.29 Hoxa3 0.29 Fam110b 0.28 Padi1 0.28 Serpina9 0.28 Igfbp4 0.28 Pdzk1ip1 0.27 Fzd1 0.27 4930546C10Rik 0.27 Foxp3 0.26 Hoxa6 0.26 Hoxa7 0.26 Edn3 0.26 Btc 0.26 Myl10 0.26 Hoxa1 0.25 Dnahc7a 0.24 Mira 0.24 Pcdhga10 0.24 Lrrc3b 0.23 Slc4a4 0.23 Rprl3 0.23 Shc2 0.22 Expi 0.22 Shisa2 0.22 Gm12709 0.22 Fbln2 0.21 Sec1 0.21 C85492 0.21 Lamc3 0.21 Dbx1 0.20 Atp1b1 0.20 Nek5 0.19 Actg2 0.19 Dntt 0.17 Nrg2 0.17 Sostdc1 0.14 Trnp1 0.14 Lama1 0.14 Rprl2 0.13 0610012H03Rik 0.12 Lrrc32 0.10

To evaluate the requirement of these ‘NK-type’ CD8 T cells in disease pathogenesis, an adoptive transfer approach was used. Cytotoxic CD8⁺ NKG2D⁺ cells transferred alone, as well as total lymph node cells, were both able to give rise to AA in five recipients, whereas lymph node populations depleted of NKG2D+ cells were unable to transfer disease (FIG. 54H). Not only are ‘NK-type’ CD8⁺ NKG2D⁺ T cells the dominant cell type in the dermal infiltrate, but, moreover, they are both necessary and sufficient for T cell mediated transfer of alopecia areata.

Using comparative genomics in order to characterize the transcriptional landscape of AA lesional skin from C3H/HeJ mice as well as humans with AA, AFFYMETRIX™ profiling was first performed of whole skin from C3H/HeJ mice with spontaneous AA versus unaffected C3H/HeJ skin (FIG. 55A, top panel and FIG. 60 ). In parallel, human skin was similarly profiled from perilesional biopsies of active disease in five AA patients versus normal controls (Tables 15-17).

TABLE 15 Differentially expressed genes in the skin of alopecic female C3H/HeJ mice vs. skin obtained from age-matched female C3H/HeJ mice without alopecia (n = 3 per group). Affy_ID GeneSymbol FC 1419762_at Ubd 135.60 1418652_at Cxcl9 74.98 1417898_a_at Gzma 56.37 1419697_at Cxcl11 53.33 1418930_at Cxcl10 37.58 1419042_at Iigp1 34.65 1418776_at Gbp8 33.06 1418126_at Ccl5 27.95 1419043_a_at Iigp1 25.36 1438676_at Gbp6 25.11 1423467_at Ms4a4b 23.27 1453196_a_at Oasl2 23.06 1419060_at Gzmb 21.74 1417141_at Igtp 21.08 1435639_at 2610528A11Rik 20.88 1423555_a_at Ifi44 20.58 1417292_at Ifi47 18.63 1425394_at BC023105 18.43 1420549_at Gbp1 17.81 1417793_at Irgm2 17.25 1435906_x_at Gbp2 15.15 1450033_a_at Stat1 14.48 1418825_at Irgm1 14.46 1444078_at Cd8a 14.23 1422812_at Cxcr6 13.98 1427747_a_at Lcn2 13.89 1420699_at Clec7a 13.82 1418240_at Gbp2 13.32 1450783_at Ifit1 13.13 1418392_a_at Gbp3 12.61 1424305_at Igj 12.53 1434380_at Gbp7 12.51 1424865_at Pyy 12.17 1422588_at Krt6b 12.07 1434046_at AA467197 11.98 1418580_at Rtp4 11.29 1419709_at Stfa3 11.04 1425156_at Gbp7 10.72 1449254_at Spp1 10.61 1440481_at Stat1 10.53 1449153_at Mmp12 10.18 1424921_at Bst2 9.80 1429947_a_at Zbp1 9.65 1456907_at Cxcl9 9.62 1421075_s_at Cyp7b1 8.74 1451777_at Ddx60 8.72 1421688_a_at Ccl1 8.65 1421074_at Cyp7b1 8.56 1419604_at Zbp1 8.51 1419714_at Cd274 8.41 1427102_at Slfn4 8.40 1424761_at Fam115c 8.26 1419135_at Ltb 8.24 1450034_at Stat1 7.98 1425832_a_at Cxcr6 7.98 1420915_at Stat1 7.67 1438037_at Herc6 7.62 1448436_a_at Irf1 7.61 1448932_at Krt16 7.12 1435710_at AI661384 7.10 1451564_at Parp14 7.08 1452614_at Bcl2l15 7.00 1417256_at Mmp13 6.88 1447541_s_at Itgae 6.79 1432026_a_at Herc6 6.70 1434372_at AW112010 6.62 1459913_at Tnfsf10 6.51 1451426_at Dhx58 6.51 1452405_x_at Trav9d-3 6.44 1429570_at Mlkl 6.41 1444064_at Samhd1 6.28 1451860_a_at Trim30a 6.23 1453080_at Apol7a 6.22 1420380_at Ccl2 6.12 1434438_at Samhd1 6.11 1450696_at Psmb9 6.06 1451537_at Chi3l1 5.99 1450165_at Slfn2 5.88 1424727_at Ccr5 5.83 1421256_at Gzmc 5.80 1448162_at Vcam1 5.80 1453913_a_at Tap2 5.79 1418191_at Usp18 5.73 1425005_at Klrc1 5.65 1439680_at Tnfsf10 5.64 1433935_at AU020206 5.51 1426113_x_at Trav9d-3 5.51 1422415_at Ang2 5.44 1460245_at Klrd1 5.41 1426971_at Uba7 5.34 1443698_at Xaf1 5.32 1418131_at Samhd1 5.26 1419591_at Gsdmc 5.20 1416897_at Parp9 5.11 1417244_a_at Irf7 5.08 1449216_at Itgae 5.06 1448632_at Psmb10 5.06 1439825_at Dtx31 5.02 1416714_at Irf8 5.00 1425917_at H28 4.99 1447621_s_at Tmem173 4.93 1425396_a_at Lck 4.88 1418536_at H2-Q7 4.84 1449025_at Ifit3 4.75 1436649_at Ikzf3 4.75 1425947_at Ifng 4.71 1417172_at Ube2l6 4.70 1438855_x_at Tnfaip2 4.70 1422962_a_at Psmb8 4.69 1421186_at Ccr2 4.66 1418204_s_at Aif1 4.61 1421911_at Stat2 4.57 1450753_at Nkg7 4.55 1450424_a_at Il18bp 4.53 1450403_at Stat2 4.52 1452565_x_at LOC641050 4.44 1441054_at Apol8 4.43 1449328_at Ly75 4.39 1422601_at Serpinb9 4.39 1422177_at Il13ra2 4.39 1426170_a_at Cd8b1 4.38 1460603_at Samd9l 4.37 1448754_at Rbp1 4.31 1417822_at D17H6S56E-5 4.28 1435208_at Dtx3l 4.26 1418649_at Egln3 4.26 1422160_at H2-T24 4.23 1456064_at AI504432 4.21 1449184_at Pglyrp1 4.19 1438498_at Zmynd15 4.19 1449195_s_at Cxcl16 4.11 1439034_at Spn 4.10 1450678_at Itgb2 4.10 1437176_at Nlrc5 4.09 1458299_s_at Nfkbie 4.07 1421262_at Lipg 4.06 1426039_a_at Alox12e 4.05 1429184_at Gvin1 4.05 1448380_at Lgals3bp 4.04 1436576_at Fam26f 4.01 1453939_x_at Gm9706 3.99 1434457_at Sp100 3.98 1425295_at Ear11 3.97 1425065_at Oas2 3.97 1449846_at Ear2 3.97 1450582_at H2-Q5 3.96 1440926_at Flt1 3.94 1457140_s_at Rassf10 3.91 1455500_at Rnf213 3.90 1426970_a_at Uba7 3.89 1448452_at Irf8 3.88 1451673_at Cd8a 3.88 1437929_at Dact2 3.86 1426774_at Parp12 3.84 1418648_at Egln3 3.82 1437811_x_at Cotl1 3.78 1428420_a_at 1200009I06Rik 3.78 1417961_a_at Trim30a 3.77 1425225_at Fcgr4 3.74 1421812_at Tapbp 3.74 1451905_a_at Mx1 3.72 1441752_at Art3 3.69 1448301_s_at Serpinb1a 3.69 1436172_at Gm20559 3.68 1418133_at Bcl3 3.67 1418655_at B4galnt1 3.66 1417821_at D17H6S56E-5 3.66 1417171_at Itk 3.63 1460227_at Timp1 3.61 1455161_at AI504432 3.61 1421228_at Ccl7 3.60 1434905_at Ndufa412 3.60 1460218_at Cd52 3.60 1460437_at Cyth4 3.54 1430005_a_at Batf2 3.53 1450188_s_at Lipg 3.53 1419698_at Cxcl11 3.52 1439790_at Serpinb9 3.51 1419413_at Ccl17 3.51 1421009_at Rsad2 3.50 1436838_x_at Cotl1 3.50 1455269_a_at Coro1a 3.50 1422828_at Cd3d 3.49 1440866_at Eif2ak2 3.47 1420437_at Ido1 3.45 1439814_at Atp8b4 3.44 1441706_at Dscaml1 3.44 1421322_a_at Irf9 3.43 1427076_at Mpeg1 3.43 1435454_a_at BC006779 3.41 1444619_x_at Psmb8 3.40 1444350_at Slfn10-ps 3.39 1425335_at Cd8a 3.38 1450484_a_at Cmpk2 3.38 1435832_at Lrrc4 3.38 1424067_at Icam1 3.37 1416318_at Serpinb1a 3.37 1445897_s_at Ifi35 3.35 1450291_s_at Ms4a4c 3.34 1416273_at Tnfaip2 3.34 1453304_s_at Ly6e 3.33 1418770_at Cd2 3.32 1434067_at AI662270 3.32 1428735_at Cd69 3.31 1424965_at Lpxn 3.31 1455393_at Cp 3.31 1429567_at Rassf10 3.30 1436905_x_at Laptm5 3.30 1428485_at Car12 3.28 1456211_at Nlrp10 3.28 1438531_at A730054J21Rik 3.26 1449175_at Gpr65 3.25 1424923_at Serpina3g 3.24 1417995_at Ptpn22 3.23 1440990_at Kif26b 3.20 1420338_at Alox15 3.18 1419300_at Flt1 3.17 1419128_at Itgax 3.16 1426872_at Fcgbp 3.15 1425336_x_at H2-K1 3.15 1448759_at Il2rb 3.14 1419178_at Cd3g 3.11 1420412_at Tnfsf10 3.11 1433465_a_at AI467606 3.11 1418203_at Pmaip1 3.11 1430208_at Krt42 3.09 1421628_at Il18r1 3.08 1417620_at Rac2 3.08 1454850_at Tbc1d10c 3.07 1439221_s_at Cd40 3.07 1438658_a_at S1pr3 3.06 1426278_at Ifi27l2a 3.06 1429692_s_at Gch1 3.04 1458148_at Nlrc3 3.03 1440927_x_at Apol11b 3.03 1436236_x_at Cotl1 3.03 1448940_at Trim21 3.02 1421217_a_at Lgals9 3.02 1451756_at Flt1 3.01 1455048_at Igsf3 3.01 1435560_at Itgal 3.01 1425719_a_at Nmi 2.99 1439956_at Ms4a6b 2.98 1436562_at Ddx58 2.98 1422632_at Ctsw 2.98 1436472_at Slfn9 2.98 1427013_at Car9 2.98 1449361_at Tbx21 2.98 1426025_s_at Laptm5 2.97 1438052_at A130071D04Rik 2.97 1426276_at Ifih1 2.97 1456890_at Ddx58 2.96 1416295_a_at Il2rg 2.94 1434873_a_at Acap1 2.93 1456103_at Pml 2.93 1418678_at Has2 2.92 1420697_at Slc15a3 2.91 1436058_at Rsad2 2.90 1425294_at Slamf8 2.90 1459151_x_at Ifi35 2.90 1418991_at Bak1 2.89 1439068_at Erap1 2.89 1424617_at Ifi35 2.89 1417189_at Psme2 2.89 1451544_at Tapbpl 2.88 1419412_at Xcl1 2.88 1419539_at Irx4 2.88 1452592_at Mgst2 2.87 1420774_a_at 4930583H14Rik 2.86 1440169_x_at Ifnar2 2.86 1420499_at Gch1 2.85 1416002_x_at Cotl1 2.85 1423754_at Ifitm3 2.84 1416942_at Erap1 2.84 1449169_at Has2 2.83 1419609_at Ccr1 2.83 1449591_at Casp4 2.81 1436598_at Icos 2.81 1427381_at Irg1 2.81 1419879_s_at Trim25 2.81 1449143_at Rtp4 2.80 1449839_at Casp3 2.78 1426599_a_at Slc2a1 2.78 1452117_a_at Fyb 2.77 1434773_a_at Slc2a1 2.77 1450378_at Tapbp 2.76 1419561_at Ccl3 2.76 1427007_at Sash3 2.76 1422706_at Pmepa1 2.76 1427892_at Myo1g 2.75 1451335_at Plac8 2.73 1425974_a_at Trim25 2.72 1426600_at Slc2a1 2.67 1418826_at Ms4a6b 2.67 1430244_at 4921509J17Rik 2.66 1457664_x_at C2 2.65 1416564_at Sox7 2.65 1433466_at AI467606 2.65 1425801_x_at Cotl1 2.64 1435945_a_at Kcnn4 2.63 1434268_at Adar 2.63 1420353_at Lta 2.63 1418741_at Itgb7 2.62 1416246_a_at Coro1a 2.62 1422280_at Gzmk 2.61 1421596_s_at H28 2.60 1415834_at Dusp6 2.60 1438364_x_at Ang4 2.60 1439819_at AU015263 2.60 1451755_a_at Apobec1 2.59 1416296_at Il2rg 2.59 1439276_at Adar 2.59 1417495_x_at Cp 2.59 1453352_at Atp10b 2.58 1437478_s_at Efhd2 2.58 1434366_x_at C1qb 2.58 1417185_at Ly6a 2.58 1443858_at Trim12c 2.58 1417494_a_at Cp 2.58 1427746_x_at H2-K1 2.57 1421034_a_at Il4ra 2.56 1422005_at Eif2ak2 2.56 1455000_at Gpr68 2.56 1415803_at Cx3cl1 2.55 1422782_s_at Tlr3 2.55 1416097_at Lrrc4 2.54 1460651_at Lat 2.53 1427325_s_at Akna 2.53 1425603_at Tmem176a 2.49 1439595_at Tcra 2.49 1424041_s_at C1s 2.49 1418718_at Cxcl16 2.48 1456061_at Gimap8 2.47 1417056_at Psme1 2.46 1418981_at Casp12 2.46 1421211_a_at Ciita 2.46 1449235_at Fasl 2.45 1449265_at Casp1 2.44 1424383_at Tmem51 2.44 1425405_a_at Adar 2.43 1425902_a_at Nfkb2 2.42 1452817_at Smyd3 2.42 1416871_at Adam8 2.42 1457917_at Lck 2.42 1449127_at Selplg 2.42 1416514_a_at Fscn1 2.42 1439773_at Ly6e 2.41 1436223_at Itgb8 2.41 1454268_a_at Cyba 2.41 1451821_a_at Sp100 2.41 1444242_at Slco2a1 2.40 1415983_at Lcp1 2.40 1418293_at Ifit2 2.39 1420671_x_at Ms4a4c 2.39 1450534_x_at H2-K1 2.39 1418077_at Trim21 2.39 1417496_at Cp 2.38 1435415_x_at Marcksl1 2.37 1451320_at Arhgap8 2.37 1427041_at BC013712 2.36 1422139_at Plau 2.36 1441768_at 9430051O21Rik 2.35 1449455_at Hck 2.35 1421358_at H2-M3 2.34 1436312_at Ikzf1 2.34 1444632_at BC064078 2.33 1450791_at Nppb 2.33 1424927_at Glipr1 2.33 1419764_at CM3l3 2.33 1426093_at Trim34a 2.33 1420515_a_at Pglyrp2 2.33 1448061_at Msr1 2.33 1418641_at Lcp2 2.32 1418842_at Hcls1 2.31 1434400_at Tgif2 2.31 1439494_at Slc5a9 2.30 1448757_at Pml 2.28 1431339_a_at Efhd2 2.28 1455581_x_at Gm20559 2.28 1437132_x_at Nedd9 2.27 1456307_s_at Adcy7 2.27 1422932_a_at Vav1 2.27 1450446_a_at Socs1 2.27 1434068_s_at AI662270 2.26 1416069_at Pfkp 2.26 1421210_at Ciita 2.26 1437785_at Adamts9 2.25 1436230_at Gprl14 2.25 1425119_at Oas1b 2.25 1449310_at Ptger2 2.25 1440721_at 5930433N17Rik 2.25 1430352_at Adamts9 2.25 1456377_x_at Limd2 2.25 1449991_at Cd244 2.25 1416051_at C2 2.25 1455132_at Rasal3 2.24 1422124_a_at Ptprc 2.24 1448576_at Il7r 2.23 1421931_at Icos 2.22 1436199_at Trim14 2.21 1446509_at Smox 2.20 1430148_at Rab19 2.20 1450170_x_at H2-D1 2.19 1439736_at 5830453J16Rik 2.19 1451567_a_at Ifi203 2.19 1433920_at Sema4c 2.18 1420913_at Slco2a1 2.18 1439148_a_at Pfkl 2.18 1452544_x_at H2-D1 2.18 1441600_at C920021A13 2.17 1422804_at Serpinb6b 2.16 1453228_at Stx11 2.15 1427682_a_at Egr2 2.15 1428378_at Zc3hav1 2.15 1430165_at Stk17b 2.15 1451321_a_at Rbm43 2.15 1457780_at Stx11 2.15 1439764_s_at Igf2bp2 2.14 1435913_at B4galnt4 2.14 1449220_at Gimap3 2.13 1456251_x_at Tspo 2.13 1422781_at Tlr3 2.13 1423135_at Thy1 2.13 1424375_s_at Gimap4 2.12 1428660_s_at Tor3a 2.12 1439012_a_at Dck 2.12 1441912_x_at C2 2.12 1424704_at Runx2 2.11 1424953_at BC021614 2.11 1431843_a_at Nfkbie 2.10 1417219_s_at Tmsb10 2.10 1448160_at Lcp1 2.10 1427352_at Krt79 2.10 1419194_s_at Gmfg 2.09 1460675_at Igsf8 2.09 1426568_at Slc2a9 2.08 1424961_at Tapbpl 2.08 1418099_at Tnfrsf1b 2.08 1422527_at H2-DMa 2.08 1426554_a_at Pgam1 2.08 1438941_x_at Ampd2 2.08 1450648_s_at H2-Ab1 2.07 1440165_at Ptprc 2.07 1437724_x_at Pitpnm1 2.07 1434364_at Map3k14 2.06 1419537_at Tfec 2.06 1425728_at Gm12185 2.06 1455966_s_at Nudt21 2.05 1419282_at Ccl12 2.05 1420404_at Cd86 2.05 1415765_at Hnrnpul2 2.05 1422511_a_at Ogfr 2.05 1425548_a_at Lst1 2.05 1427683_at Egr2 2.05 1453757_at Herc6 2.04 1435331_at Pyhin1 2.04 1419119_at Hcst 2.03 1458229_at Robo2 2.03 1422808_s_at Dock2 2.03 1437213_at Nudt21 2.02 1429831_at Pik3ap1 2.01 1439808_at Ipcef1 2.01 1428891_at Parm1 2.01 1427765_a_at Tcrb-J 2.01 1426133_a_at Mitd1 2.00 1425016_at Ephb2 2.00 1422562_at Rrad 2.00 1418110_a_at Inpp5d 2.00 1420272_at Samhd1 2.00 1417928_at Pdlim4 2.00 1460220_a_at Csf1 2.00 1451828_a_at Acsl4 2.00 1435672_at 3830612M24 1.99 1453181_x_at Plscr1 1.99 1428767_at Gsdmd 1.99 1436861_at Il7 1.99 1437270_a_at Clcf1 1.99 1419202_at Cst7 1.98 1435529_at Gm14446 1.98 1419810_x_at Arhgap9 1.98 1429525_s_at Myo1f 1.98 1437308_s_at F2r 1.98 1419676_at Mx2 1.98 1455019_x_at Ckap4 1.98 1438948_x_at Tspo 1.97 1448618_at Mvp 1.97 1436365_at Zbtb7c 1.97 1418464_at Matn4 1.97 1439093_at Hspa4l 1.97 1435193_at A230050P20Rik 1.97 1439065_x_at Gm13152 1.97 1423306_at 2010002N04Rik 1.97 1430658_a_at Gsdma2 1.97 1419469_at Gnb4 1.96 1426725_s_at Ets1 1.96 1417288_at Plekha2 1.96 1456586_x_at Mvp 1.96 1426415_a_at Trim25 1.95 1437249_at Skap1 1.95 1433517_at Myeov2 1.95 1425374_at Oas3 1.95 1425681_a_at Prnd 1.95 1436659_at Dclk1 1.94 1446326_at Col1a2 1.94 1435596_at Pion 1.94 1416492_at Ccne1 1.94 1422701_at Zap70 1.93 1435517_x_at Ralb 1.93 1436902_x_at Tmsb10 1.93 1425166_at Rbl1 1.93 1421207_at Lif 1.93 1460134_at 9330175E14Rik 1.93 1420273_x_at Samhd1 1.93 1443621_at Xaf1 1.92 1425078_x_at C130026I21Rik 1.92 1427994_at Cd300lf 1.91 1456545_at Il18rap 1.91 1418346_at Insl6 1.91 1428372_at St5 1.91 1417045_at Bid 1.91 1433699_at Tnfaip3 1.91 1440275_at Runx3 1.91 1460253_at Cmtm7 1.91 1421188_at Ccr2 1.90 1427911_at Tmem173 1.90 1420653_at Tgfb1 1.90 1437643_at Cenpj 1.90 1443687_x_at H2-DMb2 1.90 1440299_at E330016A19Rik 1.90 1425151_a_at Noxo1 1.90 1441097_at Vangl1 1.90 1449988_at Gimap1 1.89 1427404_x_at Gm5506 1.89 1425617_at Dhx9 1.88 1426926_at Plcg2 1.88 1424037_at Itpka 1.88 1449925_at Cxcr3 1.88 1435394_s_at Rhoc 1.87 1453392_at Ttc39b 1.87 1449473_s_at Cd40 1.87 1419186_a_at St8sia4 1.86 1455898_x_at Slc2a3 1.86 1430447_a_at Lair1 1.86 1418353_at Cd5 1.85 1449425_at Wnt2 1.85 1439114_at Ddx60 1.85 1425084_at Gimap7 1.85 1425819_at Zbtb7c 1.85 1416001_a_at Cotl1 1.84 1422704_at Gyk 1.84 1445452_at Traf1 1.84 1438852_x_at Mcm6 1.84 1436423_at Themis 1.84 1417599_at Cd276 1.84 1421930_at Icos 1.84 1421814_at Msn 1.83 1419132_at Tlr2 1.83 1448568_a_at Slc20a1 1.83 1423602_at Traf1 1.83 1451931_x_at H2-L 1.82 1456836_at Itk 1.82 1453129_a_at Rgs12 1.82 1426454_at Arhgdib 1.82 1451385_at Fam162a 1.82 1419470_at Gnb4 1.82 1435697_a_at Cytip 1.82 1424214_at Parm1 1.81 1458524_at Fndc3a 1.81 1455051_at Rnf31 1.81 1452163_at Ets1 1.81 1448328_at Sh3bp2 1.80 1456700_x_at Marcks 1.80 1451784_x_at H2-D1 1.80 1437606_at Btbd19 1.80 1439476_at Dsg2 1.80 1440878_at Runx1 1.79 1453472_a_at Slamf7 1.79 1434653_at Ptk2b 1.79 1436708_x_at Mcm4 1.79 1454592_at 9430012M22Rik 1.79 1433954_at 4632419I22Rik 1.78 1426315_a_at 6330416G13Rik 1.78 1451289_at Dclk1 1.78 1443686_at H2-DMb2 1.78 1449903_at Crtam 1.78 1418090_at Plvap 1.78 1460250_at Sostdc1 1.77 1435749_at Gda 1.77 1450269_a_at Pfkl 1.77 1438910_a_at Stom 1.77 1437868_at Fam46a 1.77 1448449_at Ripk3 1.77 1424496_at 5133401N09Rik 1.77 1438854_x_at Pitpnm1 1.77 1424930_s_at Fam83f 1.77 1460378_a_at Tes 1.77 1448914_a_at Csf1 1.76 1448659_at Casp7 1.76 1427689_a_at Tnip1 1.76 1428242_at Hmha1 1.76 1455975_x_at Rnf114 1.76 1453076_at Batf3 1.76 1419125_at Ptpn18 1.76 1424524_at Dram1 1.76 1453202_at E330016A19Rik 1.76 1428346_at Trafd1 1.76 1451987_at Arrb2 1.76 1417790_at Dok1 1.76 1418244_at Naa20 1.75 1416226_at Arpc1b 1.75 1438634_x_at Lasp1 1.75 1419209_at Cxcl1 1.75 1451363_a_at Dennd2d 1.75 1430622_at 4833423F13Rik 1.74 1447788_s_at Tspyl3 1.74 1456310_a_at 2610002J02Rik 1.74 1455251_at Itga1 1.74 1416750_at Sigmar1 1.74 1421830_at Ak4 1.74 1417346_at Pycard 1.74 1436958_x_at Tpm3 1.74 1434069_at Prex1 1.74 1440249_at Aknad1 1.74 1442849_at Lrp1 1.74 1451174_at Lrrc33 1.73 1451411_at Gprc5b 1.73 1460469_at Tnfrsf9 1.73 1452661_at Tfrc 1.73 1430534_at Rnase6 1.73 1456028_x_at Marcks 1.73 1448731_at Il10ra 1.73 1418257_at Slc12a7 1.73 1420170_at Myh9 1.72 1424032_at Hvcn1 1.72 1435748_at Gda 1.72 1451285_at Fus 1.72 1454607_s_at Psat1 1.72 1434089_at Synpo 1.71 1421525_a_at Naip5 1.71 1458415_at Clec2e 1.71 1419721_at Niacr1 1.71 1453328_at 2700008G24Rik 1.71 1435337_at Tshz3 1.71 1456011_x_at Acaa1a 1.71 1423986_a_at Shisa5 1.71 1427386_at Arhgef16 1.70 1443123_at Tanc2 1.70 1418674_at Osmr 1.70 1445588_at Ankrd44 1.70 1424552_at Casp8 1.70 1439261_x_at Mitd1 1.70 1452428_a_at B2m 1.70 1418045_at Inpp1 1.70 1441317_x_at Jakmip1 1.70 1436589_x_at Prkd2 1.70 1437503_a_at Shisa5 1.70 1421578_at Ccl4 1.70 1435227_at Bcl11b 1.70 1436177_at Plekha2 1.70 1457644_s_at Cxcl1 1.70 1429527_a_at Plscr1 1.70 1433531_at Acsl4 1.69 1420731_a_at Csrp2 1.69 1438097_at Rab20 1.69 1426239_s_at Arrb2 1.69 1427182_s_at D18Ertd653e 1.69 1435338_at Cdk6 1.69 1433885_at Iqgap2 1.69 1450852_s_at F2r 1.69 1446507_at Kif24 1.69 1450456_at Il21r 1.69 1418612_at Slfn1 1.68 1438974_x_at Pitpnm1 1.68 1439030_at Gmppb 1.68 1416619_at 4632428N05Rik 1.68 1441880_x_at Tmem149 1.68 1448797_at Elk3 1.68 1443794_x_at Noc4l 1.68 1452289_a_at Rnf135 1.68 1450918_s_at Src 1.67 1420955_at Vsnl1 1.67 1418751_at Sit1 1.67 1447568_at Gatc 1.67 1460700_at Stat3 1.67 1419911_at Coro1c 1.66 1452966_at Bcl11b 1.66 1421550_a_at Trim34a 1.66 1423686_a_at Prr13 1.66 1455287_at Cdk6 1.66 1439422_a_at Fam132a 1.66 1416935_at Trpv2 1.66 1436183_at Zc3hav1 1.66 1451314_a_at Vcam1 1.66 1417300_at Smpdl3b 1.66 1419097_a_at Stom 1.66 1446939_at Trim12a 1.65 1423411_at Rbm47 1.65 1450355_a_at Capg 1.65 1436220_at Zfp287 1.65 1429742_at Rcbtb2 1.65 1455221_at Abcg1 1.65 1438934_x_at Sema4a 1.65 1452836_at Lpin2 1.65 1427874_at Rnf114 1.64 1452425_at Tnfrsf14 1.64 1422303_a_at Tnfrsf18 1.64 1418396_at Gpsm3 1.64 1451462_a_at Ifnar2 1.64 1447711_x_at 4933412E12Rik 1.64 1454757_s_at Ifi27l1 1.64 1424246_a_at Tes 1.64 1459872_x_at H2-DMa 1.64 1424304_at Tpcn2 1.64 1416695_at Tspo 1.64 1451475_at Plxnd1 1.64 1428727_at Cep192 1.64 1440779_s_at Slc5a9 1.63 1433451_at Cdk5r1 1.63 1450531_at H2-Bl 1.63 1458601_at 8030447M02Rik 1.63 1426739_at Donson 1.63 1423350_at Socs5 1.63 1456467_s_at Nlk 1.63 1447803_x_at Capg 1.63 1453201_at Rassf10 1.63 1438148_at Cxcl3 1.63 1444197_at Rinl 1.63 1428859_at Paox 1.63 1455656_at Btla 1.63 1455080_at Ppp1r16b 1.63 1424857_a_at Trim34a 1.62 1450140_a_at Cdkn2a 1.62 1438095_x_at Noc4l 1.62 1460231_at Irf5 1.62 1426318_at Serpinb1b 1.62 1427348_at Zc3h12a 1.62 1451862_a_at Prf1 1.62 1442944_at C76555 1.62 1420635_a_at Tcirg1 1.62 1435143_at Elk3 1.62 1449623_at Txnrd3 1.61 1458345_s_at Colec11 1.61 1423812_s_at Vopp1 1.61 1417561_at Apoc1 1.61 1455818_at 4930427A07Rik 1.60 1426757_at Ampd2 1.60 1418265_s_at Irf2 1.60 1434185_at Acaca 1.60 1437327_x_at Enoph1 1.60 1456240_x_at Cdca4 1.60 1443086_at Alcam 1.60 1442018_at Btg1 1.60 1418686_at Oas1c 1.60 1441843_s_at 5230400M03Rik 1.60 1443882_at Slc26a2 1.60 1420909_at Vegfa 1.60 1439587_at Lemd3 1.60 1433169_at 5830456J23Rik 1.60 1437451_at Ecscr 1.59 1436074_at Nfkbid 1.59 1437789_at Birc6 1.59 1450716_at Adamts1 1.59 1438828_at Rapgef6 1.59 1453064_at Etaa1 1.59 1439047_s_at Recql 1.59 1435316_at Psma6 1.59 1428484_at Osbpl3 1.59 1417627_a_at Limk1 1.59 1459884_at Cox7c 1.59 1455660_at Csf2rb 1.59 1448511_at Ptprcap 1.59 1440245_at Ccdc88b 1.59 1418587_at Traf3 1.59 1424339_at Oasl1 1.58 1416166_a_at Prdx4 1.58 1427095_at Cdcp1 1.58 1456288_at Slfn5 1.58 1452239_at Gt(ROSA)26Sor 1.58 1449516_a_at Rgs3 1.58 1424829_at A830007P12Rik 1.58 1454402_at Zfp942 1.58 1434139_at Parp11 1.58 1428583_at Nufip2 1.58 1451097_at Vasp 1.58 1458595_at Intu 1.58 1457550_at 9530059O14Rik 1.58 1419530_at Il12b 1.58 1447762_x_at Car12 1.58 1417588_at Galnt3 1.58 1437052_s_at Slc2a3 1.58 1435084_at C730049O14Rik 1.58 1419235_s_at Helb 1.57 1440147_at Lgi2 1.57 1460347_at Krt14 1.57 1452991_at Chd2 1.57 1429758_at 1700017B05Rik 1.57 1449508_at Il27ra 1.57 1416087_at Ap1s1 1.57 1438673_at Slc4a7 1.57 1438750_at Atrx 1.57 1423869_s_at Txnrd3 1.57 1420692_at Il2ra 1.57 1449227_at Ch25h 1.57 1425241_a_at Wsb1 1.57 1450387_s_at Ak4 1.56 1427164_at Il13ra1 1.56 1455084_x_at Shmt2 1.56 1452169_a_at Dgkz 1.56 1420505_a_at Stxbp1 1.56 1455839_at Uggt1 1.56 1428118_at Lingo1 1.56 1427539_a_at Zwint 1.56 1418271_at Bhlhe22 1.56 1447851_x_at Atp10a 1.56 1452837_at Lpin2 1.56 1420171_s_at Myh9 1.55 1439253_x_at Prelid1 1.55 1452803_at Glipr2 1.55 1456914_at Slc16a4 1.55 1448202_x_at Prelid1 1.55 1452391_at Cxadr 1.55 1435595_at 1810011O10Rik 1.55 1452903_at 6230427J02Rik 1.55 1451110_at Egln1 1.55 1435564_at Neurl1b 1.55 1422489_at Mogs 1.55 1452337_at 4930427A07Rik 1.54 1459983_at Impa2 1.54 1432997_at 5830462P14Rik 1.54 1457304_at D13Ertd787e 1.54 1417955_at Ccdc71 1.54 1428034_a_at Tnfrsf9 1.54 1439455_x_at Capza1 1.54 1451608_a_at Tspan33 1.54 1417878_at E2f1 1.54 1448894_at Akr1b8 1.54 1455067_at Psd4 1.53 1457410_at Arhgap5 1.53 1436848_x_at Impa1 1.53 1436395_at Card6 1.53 1436729_at Afap1 1.53 1437185_s_at Tmsb10 1.53 1428018_a_at AF251705 1.53 1431528_at 5830427D02Rik 1.53 1425760_a_at Pitpnm1 1.53 1418492_at Grem2 1.53 1445201_at Zfp53 1.53 1459894_at Iqgap2 1.53 1438154_x_at 2610002J02Rik 1.53 1434573_at Traf3ip3 1.53 1433739_at Nol10 1.53 1434070_at Jag1 1.52 1423826_at Noc4l 1.52 1416875_at Parvg 1.52 1447792_x_at Gpr174 1.52 1417128_at Plekho1 1.52 1427260_a_at Tpm3 1.52 1416379_at Panx1 1.52 1437239_x_at Phc2 1.52 1451415_at 1810011O10Rik 1.52 1448713_at Stat4 1.52 1421937_at Dapp1 1.51 1433748_at Zdhhc18 1.51 1454350_at Intu 1.51 1429128_x_at Nfkb2 1.51 1418004_a_at Tmem176b 1.51 1423154_at BC005537 1.51 1426677_at Flna 1.51 1448600_s_at Vav3 1.51 1457539_at D10Ertd709e 1.51 1420914_at Slco2a1 1.51 1429247_at Anxa6 1.51 1452411_at Lrrc1 1.51 1449619_s_at Arhgap9 1.51 1420351_at Tnfrsf4 1.51 1437242_at Ttll12 1.51 1439444_x_at Tmed10 1.50 1448933_at Pcdhb17 1.50 1434219_at Stim2 1.50 1455428_at Fam53b 1.50 1419226_at Cd96 1.50 1435169_at A930001N09Rik 0.67 1451348_at Deptor 0.67 1425411_at Arl4a 0.67 1428864_at Dusp8 0.67 1426522_at Hadhb 0.67 1417312_at Dkk3 0.67 1418190_at Pon1 0.67 1454078_a_at Gal3st1 0.66 1425145_at Il1rl1 0.66 1417421_at S100a1 0.66 1460192_at Osbpl1a 0.66 1419963_at Deptor 0.66 1415743_at Hdac5 0.66 1435559_at Myo6 0.66 1450395_at Slc22a5 0.66 1428469_a_at Dzip1 0.66 1429884_at Srgap2 0.66 1450971_at Gadd45b 0.66 1436344_at C2cd2 0.66 1418921_at Cadm3 0.65 1421471_at Npy1r 0.65 1453571_at Deptor 0.65 1452283_at Rassf8 0.65 1416268_at Ets2 0.65 1423364_a_at Aktip 0.65 1453069_at Pik3cb 0.65 1422838_at Kcnu1 0.65 1433977_at Hs3st3b1 0.65 1426010_a_at Epb4.1l3 0.65 1455029_at Kif21a 0.65 1460406_at Pls1 0.65 1434581_at 2410066E13Rik 0.65 1422740_at Tnfrsf21 0.65 1435396_at Stxbp6 0.65 1419688_at Gpc6 0.65 1418658_at Fam82b 0.65 1419457_at Rgnef 0.65 1443904_at Fads6 0.65 1425186_at Lmbrd1 0.65 1450286_at Npr3 0.65 1425037_at Fgd4 0.64 1437871_at Pgm5 0.64 1416774_at Wee1 0.64 1458129_at Rora 0.64 1433460_at Ttc7b 0.64 1451200_at Kif1b 0.64 1448320_at Stim1 0.64 1452309_at Cgnl1 0.64 1454824_s_at Mtus1 0.64 1415981_at Herpud2 0.64 1419505_a_at Ggps1 0.64 1457686_at Akap17b 0.63 1437149_at Slc6a6 0.63 1437661_at AU021092 0.63 1443906_at Cd55 0.63 1451714_a_at Map2k3 0.63 1435693_at Mall 0.63 1451563_at Emr4 0.63 1452333_at Smarca2 0.63 1428835_at Myh14 0.63 1433711_s_at Sesn1 0.63 1448669_at Dkk3 0.63 1416778_at Sdpr 0.63 1449082_at Mfap5 0.63 1425891_a_at Grtp1 0.63 1418250_at Arl4d 0.63 1458882_at Serpinb8 0.62 1418714_at Dusp8 0.62 1431216_s_at Dnajc6 0.62 1426208_x_at Plagl1 0.62 1425138_at Guca1b 0.62 1425318_a_at Tmem116 0.62 1423176_at Tob1 0.62 1425515_at Pik3r1 0.62 1455665_at Lonrf1 0.62 1426576_at Sgms1 0.62 1440559_at Hmga2-ps1 0.62 1417545_at Trpv4 0.62 1418954_at Camkk1 0.62 1424701_at Pcdh20 0.62 1454881_s_at Upk3b 0.62 1419378_a_at Fxyd2 0.62 1448926_at Hoxa5 0.62 1437731_at C2cd2 0.62 1436501_at Mtus1 0.61 1450110_at Adh7 0.61 1417013_at Hspb8 0.61 1443969_at Irs2 0.61 1450206_at Dlc1 0.61 1428774_at Gpc6 0.61 1422904_at Fmo2 0.61 1425321_a_at Clmn 0.61 1426743_at Appl2 0.61 1427945_at Dpyd 0.61 1428321_at Eml1 0.60 1431226_a_at Fndc4 0.60 1421848_at Slc22a5 0.60 1428731_at Usp54 0.60 1456939_at Fam154b 0.60 1424408_at Lims2 0.60 1426139_a_at Ccrl1 0.60 1426332_a_at Cldn3 0.60 1416823_a_at Osbpl1a 0.60 1423994_at Kif1b 0.60 1415904_at Lpl 0.60 1429656_at Rhobtb1 0.60 1421158_at Cgnl1 0.60 1456150_at Jhdm1d 0.60 1454604_s_at Tspan12 0.59 1434307_at Tmem64 0.59 1431188_a_at Tom1 0.59 1416926_at Trp53inp1 0.59 1423447_at Clpx 0.59 1451782_a_at Slc29a1 0.59 1423174_a_at Pard6b 0.59 1436293_x_at Ildr2 0.59 1444073_at Maf 0.59 1447462_at D7Wsu130e 0.59 1424280_at Mospd1 0.59 1451270_at Dusp18 0.59 1451207_at Micu1 0.59 1455056_at Lmo7 0.59 1437409_s_at Gpr126 0.59 1436988_at Ism1 0.58 1420718_at Odz2 0.58 1425456_a_at Map2k3 0.58 1450318_a_at P2ry2 0.58 1436853_a_at Snca 0.58 1424437_s_at Abcg4 0.58 1426285_at Lama2 0.58 1417962_s_at Ghr 0.58 1455340_at Dennd5b 0.58 1415874_at Spry1 0.58 1435105_at Rnf208 0.58 1428240_at Nrxn1 0.58 1425906_a_at Sema3e 0.58 1454926_at Sphkap 0.58 1416316_at Slc27a2 0.58 1434917_at Cobl 0.57 1429778_at Optn 0.57 1436350_at Fam171b 0.57 1452207_at Cited2 0.57 1428547_at Nt5e 0.57 1426263_at Cadm4 0.57 1429036_at Otop3 0.57 1435763_at Tbc1d16 0.57 1420942_s_at Rgs5 0.57 1450974_at Timp4 0.57 1434639_at Klhl29 0.56 1421346_a_at Slc6a6 0.56 1433742_at Kank1 0.56 1435207_at Dixdc1 0.56 1450838_x_at Rpl37 0.56 1457671_at Homer2 0.56 1422008_a_at Aqp3 0.56 1415864_at Bpgm 0.56 1437181_at Peli2 0.56 1416072_at Cd34 0.56 1460514_s_at Ascl2 0.56 1420941_at Rgs5 0.56 1423966_at Cd99l2 0.55 1435435_at Cttnbp2 0.55 1428336_at Agpat4 0.55 1419458_at Rgnef 0.55 1424567_at Tspan2 0.55 1419491_at Defb1 0.55 1415865_s_at Bpgm 0.55 1422667_at Krt15 0.55 1417355_at Peg3 0.55 1439478_at Acot2 0.55 1450460_at Aqp3 0.55 1426510_at Sccpdh 0.55 1422996_at Acot2 0.55 1433454_at Abtb2 0.55 1436640_x_at Agpat4 0.55 1453008_at Trnp1 0.55 1424392_at Adhfe1 0.54 1417332_at Rfx2 0.54 1419197_x_at Hamp 0.54 1429001_at Pir 0.54 1438496_a_at Ddx26b 0.54 1454699_at Sesn1 0.54 1437217_at Ankrd6 0.54 1418412_at Tpd52l1 0.54 1424747_at Kif1c 0.54 1454646_at Tcp11l2 0.54 1421040_a_at Gsta2 0.53 1434326_x_at Coro2b 0.53 1417027_at Trim2 0.53 1460242_at Cd55 0.53 1453119_at Otud1 0.53 1420688_a_at Sgce 0.53 1450119_at Dst 0.53 1448119_at Bpgm 0.53 1456481_at Esyt3 0.53 1422168_a_at Bdnf 0.53 1434542_at Gpt2 0.52 1422905_s_at Fmo2 0.52 1425809_at Fabp4 0.52 1439549_at Prrg3 0.52 1448416_at Mgp 0.52 1437197_at Sorbs2 0.52 1422619_at Ppap2a 0.52 1436737_a_at Sorbs1 0.52 1422620_s_at Ppap2a 0.52 1427378_at Krt75 0.52 1416779_at Sdpr 0.51 1451501_a_at Ghr 0.51 1423506_a_at Nnat 0.51 1422974_at Nt5e 0.51 1425761_a_at Nfatc1 0.51 1458268_s_at Igfbp3 0.51 1460241_a_at St3gal5 0.50 1417028_a_at Trim2 0.50 1456397_at Cdh4 0.50 1434156_at Rab11fip4 0.50 1429024_at Rbm20 0.50 1441254_at Pard3b 0.50 1419289_a_at Syngr1 0.50 1422112_at Ccbp2 0.50 1444559_at Phtf2 0.50 1451410_a_at Crip3 0.50 1420017_at Tspan8 0.50 1416194_at Cyp4b1 0.50 1434013_at Ablim3 0.49 1419706_a_at Akap12 0.49 1451762_a_at Kif1b 0.49 1427371_at Abca8a 0.49 1424007_at Gdf10 0.49 1424155_at Fabp4 0.48 1428357_at 2610019F03Rik 0.48 1454654_at Dirc2 0.48 1456796_at Snai3 0.48 1417702_a_at Hnmt 0.48 1450738_at Kif21a 0.48 1422007_at Aqp3 0.48 1417014_at Hspb8 0.47 1438422_at Lrrc20 0.47 1417029_a_at Trim2 0.47 1417673_at Grb14 0.47 1428471_at Sorbs1 0.47 1439117_at Clmn 0.47 1427946_s_at Dpyd 0.46 1418317_at Lhx2 0.46 1424367_a_at Homer2 0.46 1454691_at Nrxn1 0.46 1419082_at Serpinb2 0.46 1434651_a_at Cldn3 0.46 1460569_x_at Cldn3 0.45 1431056_a_at Lpl 0.45 1443832_s_at Sdpr 0.45 1451701_x_at Cldn3 0.45 1419490_at Fam19a5 0.45 1428792_at Bcas1 0.45 1419492_s_at Defb1 0.45 1424393_s_at Adhfe1 0.44 1448978_at Ngef 0.44 1429344_at 9.13E+15 0.44 1437637_at Phtf2 0.44 1425826_a_at Sorbs1 0.44 1451620_at C1ql3 0.44 1450759_at Bmp6 0.43 1433638_s_at Hoxd8 0.43 1415996_at Txnip 0.43 1448551_a_at Trim2 0.43 1426981_at Pcsk6 0.43 1427673_a_at Sema3e 0.43 1451527_at Pcolce2 0.43 1434877_at Nptx1 0.42 1453435_a_at Fmo2 0.42 1449621_s_at Thsd1 0.42 1431805_a_at Rhpn2 0.42 1449824_at Prg4 0.41 1416129_at Errfi1 0.41 1434628_a_at Rhpn2 0.41 1455214_at Mitf 0.41 1427345_a_at Sult1a1 0.41 1418205_at Thsd1 0.40 1419414_at Gng13 0.40 1437840_s_at D2hgdh 0.40 1423635_at Bmp2 0.40 1423679_at 2810432L12Rik 0.39 1418063_at Kera 0.39 1434667_at Col8a2 0.39 1424649_a_at Tspan8 0.39 1446498_at Il20ra 0.39 1441918_x_at Serpinb6e 0.39 1415997_at Txnip 0.39 1431833_a_at Hmgcs2 0.38 1418589_a_at Mlf1 0.38 1419717_at Sema3e 0.38 1429166_s_at Clmn 0.38 1435459_at Fmo2 0.38 1434669_at Ralgps1 0.38 1431099_at Hoxd8 0.38 1434657_at Gls 0.37 1418595_at Plin4 0.36 1434474_at Abca5 0.36 1417168_a_at Usp2 0.36 1427029_at Htra3 0.35 1456895_at Cd209b 0.34 1416225_at Adh1 0.34 1454969_at Lypd6 0.34 1456741_s_at Gpm6a 0.34 1442226_at Sema3e 0.33 1451478_at Angptl7 0.33 1418706_at Slc38a3 0.32 1436279_at Slc26a7 0.32 1460244_at Upb1 0.32 1459737_s_at Ttr 0.31 1419816_s_at Errfi1 0.31 1417765_a_at Amy1 0.30 1446681_at BB086117 0.30 1433837_at 8430408G22Rik 0.30 1454965_at Fam171b 0.29 1433596_at Dnajc6 0.29 1425357_a_at Grem1 0.29 1455913_x_at Ttr 0.29 1433836_a_at 8430408G22Rik 0.28 1451382_at Chac1 0.28 1454608_x_at Ttr 0.26 1433877_at Fam46b 0.26 1425281_a_at Tsc22d3 0.25 1451535_at Il31ra 0.24 1423405_at Timp4 0.23 1435184_at Npr3 0.23 1437665_at Il22ra2 0.22 1456512_at Pdzrn4 0.22 1417788_at Sncg 0.21 1460012_at Wfdc3 0.20 1452543_a_at Scgb1a1 0.19 1419332_at Egfl6 0.19 1436643_x_at Hamp2 0.18 1453327_at Krt24 0.17 1456487_at Adcy1 0.14 1449545_at Fgf18 0.12

By comparing the corresponding lists of genes identified as differentially expressed to each other (FIG. 55A, bottom panel), three striking gene expression signatures were uncovered in this analysis. First, many of the upregulated genes in alopecic skin of both species were IFN-response genes, including the IFN-inducible chemokines CXCL 9,10 and 11¹¹ (FIG. 55A, FIG. 60 ). Secondly, several key CTL-specific transcripts were identified, including CD8A and granzymes A and B (FIG. 55A).

In turn, using CTL effectors, the small molecule JAK3 inhibitor, tofacitinib (JAK3>JAK1>>JAK2 selectivity)¹⁸, was shown to block IL-15 triggered pSTAT3 activation (FIG. 55B), and inhibit CTL effector functions, including dermal sheath cell cytotoxicity (FIG. 55C), and granzyme B/IFNγ production (FIG. 55D). Thus, hair follicle production of IL-15 provides a cytokine axis that can be interrupted pharmacologically by inhibiting its signaling in immune effectors.

Next, it was determined whether the effects of IL-15 blockade could be recapitulated by intervening downstream using small molecule inhibitors of JAK3. Using tofacitinib, systemic administration of JAK3i (FIG. 56 ) treatment prevented the development of AA and the expansion of CD8⁺ NKG2D⁺ T cells in all grafted recipients. Skin of mice treated with the protein tyrosine kinase inhibitor (PTKi) showed no histological signs of inflammation and prevented the emergence of the skin ALADIN (Alopecia Areata Disease Activity Index) transcriptional signature and Gene Expression Dynamic Index (GEDI) analysis in all mice (FIG. 56 ).

To evaluate the efficacy of JAK3i in the clinical context of AA, it was determined whether systemic JAK3i treatment could reverse established disease by initiating therapy 7 weeks after grafting, a time point at which all mice had developed extensive AA. Importantly, systemic therapy induced hair regrowth all over the body, and likewise eliminated the expansion of CD8⁺ NKG2D⁺ T cells and reversed histological markers of disease (FIG. 61 ), an effect that persisted 2-3 months after the cessation of treatment.

Finally, to test a more clinically convenient route of delivery, it was determined whether topical administration of PTKis could reverse long-standing alopecia areata with similar kinetics as systemic delivery. In established disease, topical tofacitinib, was highly effective in reversing disease in treated lesions (applied to back skin), and complete hair regrowth was observed within 12 weeks following topical therapy (FIG. 57A-B). Topical therapy was associated with the disappearance of CD8⁺ NKG2D⁺ T cells in the treated skin (FIG. 57C) and the reversal of histological markers of disease (FIG. 57D), as well as the normalization of the ALADIN transcriptional signature (FIG. 57E), and the GEDI index in all treated mice (FIG. 57F). Notably, untreated areas on the abdomen remained alopecic (FIG. 57A, middle panels), demonstrating that topical therapy was locally effective and therapeutic effects were not the result of systemic absorption. Further, these effects were durable 2-3 months after the cessation of treatment (FIG. 57A, right panels).

Taken together, the data identify CD8⁺ NKG2D⁺ T cells as the immune cellular effectors responsible for autoimmune attack of the hair follicle and provide support for a model of AA pathogenesis.

Importantly, these common mechanistic underpinnings were first revealed by in the GWAS study, which placed AA squarely among this group of allied autoimmune diseases involving NKG2DL mediated recruitment of CD8 T cell effectors, including type I diabetes^(23,24), celiac disease^(16,25) and rheumatoid arthritis²⁶. These pathways can be interrupted by small molecule inhibitors of the IL-15 downstream effector JAK kinases, the latter being particularly appealing as a topical therapeutic approach in this cutaneous disease. FDA-approved JAK inhibitors were used to show the therapeutic effect, arguing for clinical evaluation in AA with these compounds or other JAK PTKis in clinical development²⁸.

In a very short time since the GWAS findings, not only have the specific subset of cytotoxic T cells that give rise to AA been identified, but they have successfully targeted them for elimination using clinically relevant, rational therapies selected on the basis of our mechanistic studies. The findings illustrate the power of GWAS studies in uncovering new disease pathways, and, coupled with translational approaches²⁹, have rapidly opened a new and unexpected avenue for intervention in AA.

Methods

Mice. 7-10 week old female C57B1.6 and C3H/HeJ mice (Jackson Laboratories, Bar Harbor, Me.) were used and maintained under specific pathogen-free conditions.

Transfer of Alopecia Areata using grafted C3H/HeJ skin. Normal-haired C3H/HeJ mice were grafted at 8 weeks of age (during the second telogen) with skin from a C3H/HeJ mouse that developed AA spontaneously, as described previously⁷. In brief, mice spontaneously affected with AA were euthanized, full thickness skin grafts of approximately 2 cm in diameter were removed and grafted to normal-haired C3H/HeJ mice. Hair loss typically began at around 4-6 weeks after grafting.

Flow cytometric analysis of skin and cutaneous lymph nodes. To make a single cell suspension of mouse skin, fat was removed from the overlying skin in cold PBS and then incubated in collagenase type I (2 mg/ml in PBS) at 32° C. for 75 minutes. After digestion the skin was minced in RPMI/10% fetal bovine serum, filtered through a 70 mM cell strainer, and centrifuged at 1100 g for 5 minutes. The pellet was resuspended in RPMI/10% fetal bovine serum, filtered through a 40 mM cell strainer and spun at 400 g for 5 mins. The pellet was resuspended in FACs buffer (PBS/5% BSA), DAPI to gate on live cells and staining antibodies (listed in Supplemental Data). Cutaneous lymph nodes were pooled minced in RPMI, filtered through a 40 mM cell strainer, centrifuged at 400 g for 5 minutes stained and analysed on a BD LSR II flow cytometer.

Transfer of T cell populations into recipient C3H/HeJ mice. For positive selection of T cell populations, lymph node cells were obtained from 5 C3H/HeJ alopecic mice, stained with anti-CD4, anti-CD8, and anti-NKG2D antibodies, then sorted (BD Influx) to obtain two fractions: CD8⁺ NKG2D⁺ T cells, and CD8⁺ NKG2D⁻ T cells. Three to five 7-week-old C3H/HeJ mice per group were injected subcutaneously with two million sorted cells of each population. For negatively selected populations, NKG2D⁺ cells were depleted by incubating total lymph node cells from 3 alopecic C3H/HeJ mice with biotinylated anti-NKG2D (CX5) and then with streptavidin-conjugated beads (Miltenyi) prior to removal on a Miltenyi magnetic column. Five million cells (either CD8/NKG2D-depleted or total lymph node cells) were suspended in 100 ul PBS and transferred into each of 5 mice by subcutaneous injection.

Prevention and Treatment Studies. For prevention studies, mice were treatment beginning the day of grafting (n=5-10 mice per group). For JAK3i experiments: mice were implanted subcutaneously with ALZET™ osmotic mini-pumps (pumps, model 2004, Durect Corporation) on the back of each mouse to deliver vehicle (poly(ethylene glycol) (PEG)300) or vehicle containing the JAK3i tofacitinib (Abmole) at 15 mg/kg/day for 12 weeks.

For topical treatment studies, grafted mice with long-standing AA (more than 8 weeks) were treated once daily for 12 weeks to affected skin on the dorsal back with vehicle (AQUAPHOR™) or vehicle containing the JAK3 inhibitor (0.5% ointment). Full-thickness skin biopsies were excised from the dorsal surface of each mouse at interim time points, and skin samples were either snap frozen in liquid nitrogen for RNA extraction or snap frozen in OCT for immunostaining. Hair status was examined twice weekly and hair growth index calculated as described³⁰.

Immunohistochemistry and immunofluorescence. 8 mM acetone-fixed frozen mouse skin sections were air-dried and stained overnight with anti-mouse Abs (see below) at 4° C. in a moist chamber. Human hair follicles were microdissected and embedded in OCT compound prior to sectioning and staining (see below).

Primary dermal sheath and lymphokine-activated killer (LAK) cell culture. Dermal sheath (DS) cells were isolated from microdissected mouse vibrissa follicles and cultured in 20% FBS DMEM with 5 ng/ml murine FGF (Pepro Tech). LAK cells were generated from splenocytes plated at 4×10⁶ in 6-well plates with 50 nM JAK3i (tofacitinib) or 50 ng/ml murine IL-15 plus 50 nM JAK3i, and incubated at 37° C. in a 5% CO₂ incubator for 96 hours.

In vitro cytotoxicity assays. Determination of specific killing of target cells was performed using CFSE-labeled DS cells at targets mixed with different ratios of effector cells incubated for 5 hours at 37° C. 5% CO2 with or without neutralizing rat anti-mouse NKG2D antibody (20 ug/ml) (Biolegend, CX5). Specific lysis of DS cells was determined flow cytometrically by measuring cell death of CFSE+ DS cells using Annexin V/7-AAD.

Gene expression analysis in human and mouse skin and T cells. Total RNA was isolated using the MIRNEASY™ Mini Kit (Qiagen Inc., Valencia, Calif., USA) with on-column DNA digestion using the RNase-free DNase set (Qiagen, Inc.). For RNAseq analysis CD3+CD8+CD44+ NKG2D+ and CD3+CD8+CD44+ NKG2D− cells were flow-sorted from lymph nodes of alopecic C3H/HeJ mice. RNA was extracted as above and prepared for RNA-seq using the TRUSEQ™ RNA Sample Prep Kit v2. Samples were sequenced on the HISEQ™ 2000 sequencer (Illumina, San Diego, Calif.) for 50 cycles. RNA-Seq files were demultiplexed by the Rockefeller University Genomics Core Facility.

For global transcriptional profiling in mouse skin, total extracted RNA was processed using the 3′ IVT EXPRESS KIT™ from AFFYMETRIX™. Resulting biotinylated cDNA samples were hybridized to the Mouse Genome 430 2.0 gene chips and subsequently washed, stained with streptavidin-phycoerythrin, and scanned on an HP GENEARRAY™ Scanner (Hewlett-Packard Company, Palo Alto, Calif.).

For human AA samples, perilesional punch biopsies from 5 patients with patchy alopecia areata who were not undergoing local or systemic treatments were collected and compared to scalp biopsies from 5 unrelated unaffected individuals.

Extracted total RNA were reverse transcribed and amplified using the OVATION™ RNA Amplification V2 kit (NuGEN Technologies, Inc., San Carlos, Calif.). Amplified cDNA was biotinylated with the ENCORE™ Biotin Module (NuGEN Technologies) and then hybridized to the U133A Plus 2.0 gene chips.

Antibodies used for flow cytometry and immunostaining. Flow cytometric analysis used the following anti-mouse antibodies: CD3 (17A2, Ebioscience), CD4 (GK1.5, BD), CD8a (53-6.7, BD), CD8a (YTS156.7.7, Biolegend), NKG2D (CX5, Ebioscience), NKG2A/C/E (clone 20d5, Ebioscience), CD44 (IM7, BD), CD45 (30-F11, BD), CD49b (Dx5, BD), CD62L (MEL-14, BD), CD69 (H1.2F3, BD), CD103 (2E7, eBioscience), IFNγ (XMG1.2, Ebioscience), Granzyme B (NGZB, eBioscience), Rae-1 (186107, R&D).

For immunohistochemical studies of mouse skin, 8 pM methanol-fixed frozen skin sections were stained with primary rat antibodies (Biolegend) including: anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), Biotin anti-MHC class I (clone 36-7.5), anti-MHC class II (clone M5/114.15.2). Biotinylated goat anti-rat IgG (Life Technologies) was used as secondary antibody. For immunofluorescence studies anti-H60 (R&D, clone 205326), anti-Pan Rae-1(R&D, clone 186107), anti-NKG2D (R&D clone 191004), anti-K71 (Abcam) primary antibody were used in immunofluorescence. ALEXA FLUOR™ 488 or ALEXA FLUOR™ 594-conjugated goat anti-Rat, donkey anti-Rabbit or donkey anti-Goat antibody was used as secondary antibody (Life Technologies).

Human hair follicles were microdissected and embedded in OCT compound prior to sectioning and staining. 8 pM methanol-fixed frozen sections were stained with CD8 (SCBT, C8/144B) followed by staining with Alexa Fluor 488 or Alexa Fluor 594-conjugated secondary antibody (Life Technologies). All images were captured with an SDRC Zeiss Exciter Confocal Microscope.

Statistical Analysis and Quality Control of Gene Expression Signatures

RNA-Seq analysis. Samples were sequenced on the HiSeq 2000 sequencer (Illumina, San Diego, Calif.) for 50 cycles. RNA-Seq files were demultiplexed by the Rockefeller University Genomics Core Facility. Quality control of the sample fastq files was performed using fastqc^(,S1). TopHat^(,S2) was used to map transcripts to the UCSC mm9 reference genome from iGenome. The RefSeq gene annotation packaged with this iGenome version of the UCSC mm9 were used. The htseq-count utility from the HTSeq package was used to convert TopHat bam files to counts that could be used as input for downstream analysis of differential expression with edgeR^(,S3). Absent genes were removed and a pseudocount of I was added in order to avoid division by zero in downstream analysis. EdgeR was used to identify differentially expressed genes using a matched pairs design with three biological replicates.

Microarray Analysis.

Quality Control, Preprocessing. For the mouse cDNA samples were hybridized to the Mouse Genome 430 2.0 gene chips and subsequently washed, stained with streptavidin-phycoerythrin, and scanned on an HP GeneArray Scanner (Hewlett-Packard Company, Palo Alto, Calif.). For the human, amplified cDNA was hybridized to the U133A 2.0 gene chips.

Quality control was performed using the affyanalysisQC package from arrayanalysis.org/. AffyanalysisQC uses R/BioConductor packages: affy, affycomp, affypdnn, affyPLM, affyQCReport, ArrayTools, bioDistm biomaRt, simpleaffy, yaqcaffy to perform QC within a single script. RMA normalization^(,S4) was performed on each experimental group separately. Batch effect correction using ComBat was required for the prevention experiments.

Microarray preprocessing was performed using BioConductor in R. Preprocessing of the three experiments, 1) spontaneous AA mice vs. normal mice, 2) prevention mice with three treatments vs placebo and sham-operated mice, and 3) treatment mice for two treatments vs. placebo were performed separately using the same pipeline. In addition to the preprocessing that was done for the mouse skin samples, Harshlight was used to correct for image defects for the human skin samples.

Identification of Gene Signatures.

Unsupervised analysis. Hierarchical clustering was performed using Cluster^(,S5) on the 363 genes from the human 5×5 and 583 genes from the spontaneous mouse 3×3 in order that met the threshold abs(log FC) >1, unadjusted p-value <=0.05. Genes were first selected that met the threshold log FC>1, and unadjusted p-value <=0.05.Genes were median centered and normalized. Spearman rank correlation was used as the similarity measure and average linkage was used to perform row (genes) and column (sample) clustering. Visualization of the hierarchical clusters was performed with java TreeView^(,S6). Gene Expression Dynamic Index (GEDI) analysis was used to visualize how “metagenes” identified with a self organizing map algorithm vary across samples^(,S7). Metagenes are clusters of genes that show similar expression patterns across samples and that are assigned to a single pixel in a two dimensional grid. Neighboring pixels demonstrate similar expression patterns to one another.

Supervised analysis. Initial analysis of differential gene expression was performed on the spontaneous mouse 3×3 and the human 5×5 data sets using limma^(,S8). A threshold of 1.5 fold change and unadjusted p-value of 0.05.

RT-PCR Validation. Predicted differentially expressed genes in human and mouse were confirmed using RT-PCR. First-strand cDNA was synthesized using a ratio of 2:1 random primers: Oligo (dT) primer and SUPERSCRIPT™ III RT (Invitrogen) according to the manufacturer's instructions. qRT-PCR was performed on an ABI 7300™ machine and analyzed with ABI Relative Quantification Study™ software (Applied Biosystems, Foster City, Calif., USA). Primers were designed according to ABI guidelines and all reactions were performed using Power SYBR™ Green PCR Master Mix (Applied Biosystems), 250 nM primers (Invitrogen) and 20 ng cDNA in a 20 L reaction volume. Primer sequences are provided in Table 16.

TABLE 16 RT-PCR Primers for mouse mRNA validation studies. Mouse RT-PCR SEQ ID SEQ ID Gene Forward Primer NO: Reverse Primer NO: Cd8a GAGACCAGAAGAT 12 GCCTGGGACATTTGC 13 TGTCGGC AAACA Cd3d ACTGTGTGGAGCT 14 CTGTACTGGGTATCTT 15 AGACTCG CACG Stat1 CGCAACTACAAAG 16 ATCCAGTTCGCTTAG 17 TCATGGC GGTCG Ifng ACGGCACAGTCAT 18 GCTGATGGCCTGATT 19 TGAAAGC GTCTT Tap1 CCTGCTTATCTTGG 20 GGTAGCACCCTCCTCT 21 ATGATGC CTT Cxcl9 ACGGAGATCAAAC 22 TTCCCCCTCTTTTGCT 23 CTGCCTA TTTT Cxcl10 ATCCACCGCTGAG 24 CCTTGAGTCCCACTCA 25 AGACATC GACC Cxcl11 TAGCCCTGGCTGC 26 ACTTTGTCGCAGCCGT 27 AATATCT TACT Irf7 GCACCCTCCTTTTC 28 GCCAAGGTGGCTGTA 29 ACTGAG GATGT Ccl5 CCCTCACCATCAT 30 GAGCACTTGCTGCTG 31 CCTCACT GTGTA H60a TGCCTGATTCTGA 32 ATTCACTGAGCACTG 33 GCCTTTTCA TCCATGTAGAT H60b AGGCCTTTTGGTC 34 ATGTTTTTTATCACCA 35 CTGCTGAAT AAATCAAGGAGT H60c AGATTTCAGTTGC 36 ACATGTGCAGCAGTG 37 TGCCTCA GTTG *Commercial Solaris real-time PCR system primers and probe sets AX-047904-01, AAX-047854-01, AX-047833-01, AX-049954-01, AX-042357-01 and AX-040917-00 (Thermo Scientific) were used for PCR of Rae-1a, Rae-1b, Rae-1c, Rae-1d, Rae-1e and GAPDH. Results are expressed as ′fold increase′ in mRNA expression.

TABLE 17 RT-PCR Primers for human mRNA validation studies. Human RT-PCR SEQ ID SEQ ID Gene NO. Forward Primer NO: Reverse Primer GZMA 38 AGATTTCTGGCATCCTCTC 39 GACCATGTAGGGTCTTGA IL15 40 TTTCAGTGCAGGGCTTCCT 41 GGGTGAACATCACTTTCCC STAT1 42 GCAGGTTCACCAGCTTTAT 43 TGAAGATTACGCTTGCTTT CXCL9 44 GTAGTGAGAAAGGGTCGC 45 AGGGCTTGGGGCAAATTG IFNG 46 TCGGTAACTGACTTGAATGTCCA 47 TCGCTTCCCTGTTTTAGCTGC

The following PCR protocol was used: step 1: 50° C. for 2 min; step 2: 95° C. for 10 min; step 3: 95° C. for 15 s; step 4: 60° C. for 1 min; repeat steps 3 and 4 for 40 cycles. All samples were run in quadruplicate for three independent runs and normalized against an endogenous internal control as indicated.

ALADIN scores. The IFN and CTL signatures were used to develop a bivariate score statistic. Individual signature IFN and CTL scores were determined following procedures used in human SLE^(,S9,S10). The sets of genes selected to comprise our IFN and CTL signatures were CD8A, GZMB, and ICOS for the CTL signature, and CXCL9, CXCL10, CXCL11, STAT1, and MX1 for the IFN signature. The scores for the prevention mice were calculated in relation to the sham mice; whereas, the scores for the topical treatment experiments were calculated relative to all the samples at week zero.

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Example 11—Effect of JAK3 Inhibitor on the Induction of Hair Growth

The JAK-STAT signaling pathway has been implicated in several developmental processes, and most recently in stem cell maintenance, activation and differentiation. In the course of the inventor's topical treatment studies using JAK3 in the C3H/HeJ AA mouse model, it was noticed that the hairs that regrew did so with two striking features that were different from systemic administration: 1) hair regrowth was very rapid; and 2) the hair coat was darkly pigmented. Without being bound by theory, in addition to the JAK3 inhibitor eliminating pathogenic T cells from the skin, the JAK3 inhibitor also has a direct effect on the hair follicle itself, for example, via an anagen-promoting effect.

The dynamics of the JAK-STAT signaling pathway were first interrogated using a targeted RT-PCR array containing readouts of JAK-STAT signaling (FIG. 63 ), and compared gene expression between the telogen vs. anagen stages of the normal hair cycle (FIGS. 64-66 ).

This data revealed that many components of JAK-STAT signaling were upregulated in telogen and downregulated in anagen phase of the normal hair cycle, indicating that in the context of the hair cycle, JAK-STAT signaling can be associated with maintaining stem cell quiescence in telogen (FIG. 66 ).

To test whether inhibition of JAK-STAT signaling could therefore trigger the telogen-to-anagen transition, a topical JAK3 inhibitor was applied to test whether anagen could be induced in normal mouse skin in telogen.

Indeed, topical administration of a JAK3 inhibitor resulted in a striking anagen induction in mouse skin in telogen, compared to vehicle alone. This was associated with marked proliferation of keratinocyte matrix cells, and the induction and growth of robust pigmented anagen hairs after 1-2 weeks (see FIGS. 69-74 ).

This observation was compared to a positive control SAG (sonic hedgehog agonist) which is known to have the same effect, and the JAK3 inhibitor was comparable in its effect on anagen induction (FIGS. 69, 73, 74 ).

Without being bound by theory, these findings indicate that blockade of JAK-STAT signaling in telogen mimics in part the molecular events of anagen initiation, and can be a useful therapeutic agent for hair growth, using topical JAK inhibitors to induce telogen hairs to re-enter anagen. 

What is claimed is:
 1. A method of treating alopecia areata in a human subject in need thereof comprising orally administering to the human subject once daily a therapeutically effective amount of LY3009104 to treat alopecia areata in the human subject.
 2. The method of claim 1, wherein the alopecia areata is in patches.
 3. The method of claim 1, wherein the alopecia areata is alopecia totalis.
 4. The method of claim 1, wherein the alopecia areata is alopecia universalis.
 5. The method of claim 1, wherein the LY3009104 is in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
 6. The method of claim 1, wherein the alopecia areata is severe alopecia areata.
 7. The method of claim 6, wherein the severe alopecia areata is in patches.
 8. The method of claim 6, wherein the severe alopecia areata is alopecia totalis.
 9. The method of claim 6, wherein the alopecia areata is alopecia universalis.
 10. The method of claim 6, wherein LY3009104 is in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
 11. A method of treating alopecia areata in a human subject in need thereof comprising orally administering to the human subject a therapeutically effective amount of LY3009104 to treat alopecia areata in the human subject.
 12. The method of claim 11, wherein the alopecia areata is in patches.
 13. The method of claim 11, wherein the alopecia areata is alopecia totalis.
 14. The method of claim 11, wherein the alopecia areata is alopecia universalis.
 15. The method of claim 11, wherein LY3009104 is in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
 16. The method of claim 11, wherein the alopecia areata is severe alopecia areata.
 17. The method of claim 16, wherein the severe alopecia areata is in patches.
 18. The method of claim 16, wherein the severe alopecia areata is alopecia totalis.
 19. The method of claim 16, wherein the alopecia areata is alopecia universalis.
 20. The method of claim 16, wherein LY3009104 is in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
 21. A method of treating severe alopecia areata in a human subject in need thereof comprising orally administering to the human subject once daily a therapeutically effective amount of LY3009104 to treat alopecia areata in the human subject, wherein LY3009104 is the only therapeutically effective agent being administered to the human subject for treating severe alopecia areata.
 22. The method of claim 21, wherein the alopecia areata is in patches.
 23. The method of claim 21, wherein the alopecia areata is alopecia totalis.
 24. The method of claim 21, wherein the alopecia areata is alopecia universalis.
 25. The method of claim 21, wherein the LY3009104 is in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. 